Current Biology 24, 2006–2011, September 8, 2014 ª2014 The Authors http://dx.doi.org/10.1016/j.cub.2014.07.

035

Report
Telomerase Activation
after Recruitment in Fission Yeast

Christine Anne Armstrong,1 Siân Rosanna Pearson,1 [18, 19]. Thus, association of telomerase with the telomere
Hanna Amelina,1 Vera Moiseeva,1 and Kazunori Tomita1,* through Est1 requires TER1. However, the possibility exists
1Chromosome Maintenance Group, UCL Cancer Institute, that telomerase is retained at telomeres through another
University College London, London WC1E 6DD, UK connection(s) because the association of telomerase with
Ccq1 can be maintained without RNA [12, 13]. To investigate
this further, the association between Trt1 and each of the
Summary proteins Ccq1, Tpz1, and Est1 was assessed by coimmuno-
precipitation (coIP) in the presence of ribonuclease (RNase)
Current models depict that telomerase recruitment equates (Figure 1A), which substantially reduced TER1 levels (Fig-
to activation. Telomeric DNA-binding proteins and the telo- ure 1B). Interestingly, whereas the association between Trt1
merase accessory proteins coordinate the recruitment of and Est1 was reduced by RNase treatment, association of
telomerase to the ends of chromosomes in a telomere Trt1 with both Ccq1 and Tpz1 was resistant to RNase treat-
length- and cell-cycle-dependent manner [1–4]. Recent ment, confirming that these interactions are not bridged
studies have demonstrated that the telomeric protein TPP1 by TER1. Given that Trt1 is not recruited to the telomere in
and its binding protein TIN2 are key proteins for both telome- ter1D and est1D mutants [13, 20], Trt1 must retain its connec-
rase recruitment and processivity in mammalian cells [5–7]. tion with the Pot1 complex via an alternative interaction after
Although the precise molecular mechanism of telomerase recruitment through the Ccq1-Est1-TER1 pathway.
recruitment has not yet been established, targeted point mu- Intriguingly, the binding domains for Est1 and Tpz1 in Ccq1
tations within the oligonucleotide/oligosaccharide-binding seem to overlap [15]. Similarly, the 14-3-3-like domain of Est1
(OB)-fold domain of TPP1 have been shown to impair telo- binds to both Ccq1 and TER1 [13]. Hence, their interactions
merase association and processivity [8–10]. In fission yeast, could be mutually exclusive. To test this hypothesis, we con-
telomerase is recruited through an interaction between the ducted yeast three-hybrid analyses, in which expression of a
telomerase subunit Est1 and Ccq1, a component of the third factor (protein or RNA) was induced by removal of methi-
Pot1-Tpz1 telomere complex (POT1-TPP1 orthologs) [11– onine from the media. Although Ccq1 and Est1 were able to
15]. Here, we demonstrate that association of telomerase interact under methionine plus conditions, removal of methio-
with telomeres does not engage activity. We describe a nine to allow coexpression of TER1 or Tpz1 disrupted the
mutation of Tpz1 that causes critical telomere shortening Ccq1-Est1 interaction (Figure 1C; Figures S1A and S1B avail-
despite telomeric accumulation of the telomerase catalytic able online). This indicates that Ccq1 cannot bind Est1 and
subunit, Trt1. Furthermore, Est1-directed telomerase asso- Tpz1 simultaneously and also that Est1 cannot simultaneously
ciation with Ccq1 is transient, and the Est1-Ccq1 interaction bind both Ccq1 and TER1. These results raise the possibility
does not remain the bridge between telomeres and telome- that in order to form the Ccq1-Est1 complex, Ccq1 and
rase. Rather, direct interaction of Trt1 with Tpz1 is critical Est1 must detach from Tpz1 and the telomerase complex,
for telomere elongation. Moreover, Ccq1, which has been respectively. Collectively, our data suggest that the Ccq1-
well characterized as a telomerase recruiter, is also required Est1 interaction does not bridge Tpz1-Ccq1 and Trt1 and pre-
for the activation of telomere-associated telomerase. Our dict an alternative association between shelterin and Trt1 after
findings reveal a layer of telomerase regulation that controls recruitment to the telomere by Est1.
activity after recruitment.
Overexpression of Tpz1 Confers Telomere Elongation
Telomere length homeostasis is in part regulated by the avail-
Results and Discussion
ability of telomeric proteins and telomerase components. To
investigate whether Est1 is involved in telomerase activation,
Temporal Association of Est1 with Ccq1 at Telomerase-
we overexpressed Est1 as well as Trt1 and Tpz1. Contrary to
Active Telomeres
the results of studies showing that hEst1A overexpression
The S. pombe telomerase complex contains a reverse-tran-
results in end-to-end chromosome fusions and shortening of
scriptase catalytic subunit (Trt1), an RNA template (TER1),
telomeres in human cells [21, 22], overexpression of Est1 in
and a telomerase-binding protein (Est1) [16–19]. The activity
S. pombe did not affect telomere length (Figures S1C and
of telomerase at telomeres is positively and negatively
S1D). However, a number of Est1 (or SMG) family proteins exist
controlled by the shelterin complex, which in S. pombe com-
in mammals, and their crucial roles in the nonsense-mediated
prises Taz1, Rap1, Poz1, Tpz1, Pot1, and Ccq1 [11]. Telome-
mRNA decay pathway make it difficult to assess their function
rase is recruited to the telomere via an interaction between
in telomerase recruitment. Intriguingly, however, we found that
Est1 and Ccq1 [13, 15], which occurs as a result of primarily
telomeres were elongated slightly by Trt1 overexpression and
Rad3 (ATR)-mediated phosphorylation of Ccq1 at threonine
profoundly by Tpz1 overexpression (Figures S1C and S1D).
93 (T93), which provides a binding site for the 14-3-3-like phos-
These findings largely recaptured similar phenomena that
pho-binding domain of Est1 [13–15]. In the current S. pombe
have been observed previously in human cell lines [23, 24].
model, the Trt1-Est1 association is maintained through TER1
The telomere elongation caused by Tpz1 overexpression is
not due to disruption of shelterin function because deletion
*Correspondence: k.tomita@ucl.ac.uk of poz1 in a strain overexpressing Tpz1 results in additive
This is an open access article under the CC BY-NC-ND license (http:// elongation of telomeres (Figure S1E). Rather, it is likely to
creativecommons.org/licenses/by-nc-nd/3.0/). stem either from promiscuous recruitment of telomerase to
Telomerase Activation after Recruitment
2007

A with telomerase [8–10]. This region has been coined the

‘‘TEL patch’’ (TPP1 glutamate [E]- and leucine [L]-rich patch)
[10]. The corresponding residues were systematically mutated
with COOH terminus HA tagging at one allele of tpz1+ in diploid
cells, and the stability of the mutant Tpz1 was determined (Fig-
ure S2A). The telomere lengths of diploid strains heterozygous
for the tpz1 mutation and the tpz1 mutant haploid offspring
were determined by Southern blot (Figures S2B and S2C).
Alanine substitution of several amino acid residues within the
corresponding region of Tpz1 (EKRI at position 74–77; and
TS at position 78–79) was found to cause shortening of telo-
meres (Figure S2C). Further mutagenesis of individual resi-
dues within this region enabled us to identify lysine 75 (K75)
and threonine 78 (T78) as the key residues required for
telomere lengthening (Figures 2A and S2C). These residues
B C appear to act in an epistatic manner because telomere short-
ening in the double-mutant tpz1-K75A-T78A was not additive.
Furthermore, it seems that the charge of the residue is impor-
tant for function because substitution of K75 with arginine did
not affect telomere length, whereas substitution with alanine
did (Figure 2A). Indeed, substitution of T78 with the negatively
charged residues aspartic acid (T78D) or glutamic acid (T78E)
resulted in defective telomere maintenance, equal to T78A.
Conversely, I77A and S79A mutations resulted in slight elon-
gation of telomeres (Figure S2C). Curiously, slow-migrating
bands were observed prominently in extracts from strains
expressing K75A, R76A, and T78A mutant forms of Tpz1.
Phosphatase treatment suggested that the shifted bands are
due to phosphorylation of the Tpz1 protein (data not shown).
In summary, the TEL patch-like region in Tpz1 can modulate
Figure 1. The Association of Est1 with Ccq1 during Telomerase Recruit- telomere length homeostasis, and the charge imparted by
ment Is Likely to Be Transient
residues K75 and T78 is critical for elongation of telomeres.
(A) Associations of Trt1 with Ccq1 and with Tpz1 are retained in the absence
To assess whether telomere shortening in the tpz1-K75A
of TER1. Whole-cell extracts (WCEs) were immunoprecipitated (IP) with
anti-PK (aPK) antibody in the presence or absence of RNase to purify mutant is caused by impaired telomerase activity, heterozy-
Ccq1, Tpz1, or Est1 complexes. The resulting immunoprecipitates were hy- gous tpz1K75A/+ diploids were sporulated, and telomere length
bridized with either anti-PK or anti-Myc. Cdc2 was used as a control for was monitored in the tpz1-K75A offspring over time. A trt1D/+
sample input. Histone H3 was used as a control for the presence of DNA. diploid strain was also sporulated, and the trt1D haploid
(B) RNase treatment reduces TER1 levels. RNA extracted from 5% of each offspring were examined side by side with the tpz1-K75A
IP sample was subjected to RT-PCR. TER1 PCR products were visualized
haploid cells. Similar to trt1D cells, tpz1-K75A haploid mutants
on a 2% agarose gel. As a control, the RT enzyme was substituted with
water (2RT). exhibited progressive shortening of telomeres with increasing
(C) Yeast three-hybrid analysis: coexpression of Tpz1 or TER1 disrupts generations (Figure 2B). However, the extent of telomere loss
Ccq1-Est1 binding. Equal amounts of cells were spotted on selection plates was smaller in tpz1-K75A mutants compared to trt1D cells.
(2His 2Ade) and a nonselective plate to control for loading (+His +Ade). Unlike trt1D cells, the shortened telomeres were maintained
Coexpression of Tpz1 or TER1 from the MET17 promoter was induced by during later generations rather than being lost completely.
removal of methionine (Met) from the media. Expression of gene products
This phenotype appears similar to that of ccq1D cells in which
is shown in Figure S1.
short telomeres are maintained by homologous recombination
[12]. However, in the case of tpz1-K75A cells, the short telo-
telomeres regardless of length, or enhanced telomerase activ- meres are maintained by telomerase because they were lost
ity. Notably, Tpz1 overexpression increased the length of the completely after further deletion of the trt1+ gene (Figure 2C).
entire population of telomeres, rather than merely increasing Furthermore, the inability to recruit telomerase due to intro-
telomere length heterogeneity (Figure S1C). Thus, our results duction of the ccq1-T93A mutation in the tpz1-K75A strain
favor the intriguing possibility that Tpz1, the ortholog of also resulted in telomere loss (Figure 2D). Thus, short telo-
mammalian TPP1, may be involved in targeted telomerase meres in tpz1-K75A mutants are maintained by impaired telo-
recruitment/retention and activation in fission yeast. merase activity.
Because Tpz1 is part of the shelterin complex and shelterin
Lysine 75 and Threonine 78 of Tpz1 Are Required for formation can negatively control telomerase activity, it is
Telomere Extension possible that telomere shortening in the tpz1-K75A mutant is
Because the structure and function of Tpz1 are conserved with occurring through enhanced suppression of telomerase activ-
that of human TPP1 (hTPP1) [11], it is tempting to speculate ity. Our current knowledge of Tpz1 protein structure and func-
that a telomerase affinity region resides in the oligonucleo- tion suggests that K75 falls within the Pot1-binding domain
tide/oligosaccharide-binding (OB) fold domain of fission yeast [11]. As such, mutation of this residue might enhance the abil-
Tpz1 as it does in hTPP1. Several core residues within the loop ity of Tpz1 to interact with Pot1, resulting in a stronger shelterin
region after the second a helix of the OB fold of hTPP1 have formation that blocks telomerase activity. Using yeast two-
been identified as responsible for the association of TPP1 hybrid analysis, we reassessed whether the OB-fold domain
Current Biology Vol 24 No 17
2008

A B Figure 2. Mutation of the OB-Fold Domain of

Tpz1 Results in Impaired Telomerase Activity
(A–D) Telomere Southern blots of genomic DNA
digested with EcoRI and hybridized with a telo-
meric probe. A slice of the EtBr-stained gel image
at 2.5 kb is shown below the blots as a loading
control. (B) Genomic DNA was harvested at
multiple intervals (as indicated) over the course
of >2 weeks after sporulation of diploid strains.

Tpz1-K75 Controls Telomerase

Activity after Recruitment
Because the OB-fold domain of hTPP1
can recruit human TERT [8], S. pombe
Trt1 may associate with, and be retained
at telomeres by, Tpz1 after recruitment
via Est1. As such, the K75A mutation
in Tpz1 might impair the stability of the
interaction between the Pot1 complex
and telomerase. To address this pos-
sibility, we examined the efficiency of
C D association between Tpz1 and Trt1 by
coIP. Surprisingly, Trt1 was enriched in
immunoprecipitates containing Tpz1-
K75A compared to wild-type (WT) Tpz1
(Figure 3A). Furthermore, chromatin
immunoprecipitation (ChIP) experi-
ments indicate that strains carrying mu-
tant tpz1-K75A exhibit slightly greater
enrichment of Trt1 at the telomere than
strains carrying the WT tpz1+ gene (Fig-
ure 3B). Interestingly, the efficiency of
association observed by coIP between
Ccq1 and Trt1 was also increased in
tpz1-K75A mutants, whereas the inter-
action between Ccq1 and Tpz1 was
not affected (Figure 3C). Thus, Trt1 is
not only recruited but also appears to
accumulate at the telomere in tpz1-
K75A mutants.
Although Tpz1-K75A associates with
Trt1, telomeres are short, implying that
of Tpz1 (residues 1–154) is required for binding to Pot1. By telomerase activity/processivity is compromised. Because
analyzing interactions with a range of Tpz1 truncation proteins, TEL patch mutations in TPP1 impair the efficiency of both
we were able to resolve the binding sites for Pot1, Ccq1, and TERT association and telomerase processivity in human cells,
Poz1 to residues 155–213, 422–490, and 479–508, respectively it is possible that a direct interaction between Tpz1 and Trt1
(Figure S3A). Thus, like mammalian TPP1, the OB-fold of within the shelterin-telomerase complex is necessary for full
S. pombe Tpz1 is not required for Pot1 binding or binding to telomerase activity after recruitment in fission yeast. To genet-
Poz1 or Ccq1. ically test this speculation, we generated strains expressing a
Mutation of taz1, rap1, and poz1 leads to disruption of Trt1-Tpz1 fusion protein. Tandem PK tags and the tpz1 open
shelterin formation and massive telomerase-dependent elon- reading frame were inserted before the stop codon of the
gation of telomeres [11, 20, 25, 26]. Mutation of tpz1+ to endogenous trt1 gene in a heterozygous tpz1D/+ diploid strain
tpz1-K75A in strains carrying deletions of the taz1, rap1, or (Figure S4A). The resulting diploid cells exhibited WT telomere
poz1 genes resulted in telomeres that were significantly longer length, as did tpz1+ haploid strains carrying the fusion protein
than those in cells carrying only the tpz1-K75A mutation. How- (Figure 4A), and the fusion protein was stably expressed (Fig-
ever, the presence of the tpz1-K75A mutation reduced the ure S4B). Dysfunction of either Trt1 or Tpz1 is known to lead to
telomere elongation seen in taz1D, rap1D, and poz1D single telomere loss [11, 16]. However, haploid tpz1D strains in which
mutants (Figure S3B). This result suggests that telomerase the endogenous trt1+ gene was replaced with the trt1-tpz1
activity is lower in the K75A mutant strain, even in the absence chimera gene retained telomeres, indicating that the two fused
of shelterin formation. Together, our results show that the proteins are functional. In fact, telomeres were slightly elon-
tpz1-K75A mutation does not affect the formation and function gated in Trt1-Tpz1 fusion strains carrying tpz1D, which might
of the shelterin complex. Rather, it directly impairs telomerase reflect loss of selective targeting of telomerase to short telo-
activity. meres (Figure 4A). Intriguingly, the telomere-shortening defect
Telomerase Activation after Recruitment
2009

A observed in tpz1-K75A strains was rescued by fusing Tpz1-

K75A with Trt1 (Figure 4B). Thus, fusion of Tpz1 with Trt1
can overcome the telomerase activity defect caused by the
tpz1-K75A mutation. OB-folds are often composed of a num-
ber of hydrophobic and positively charged residues that are
important for mediating interactions with DNA [27]. It is
possible that charge is also important for interaction of the
OB-fold with the Trt1 complex, and alteration of this charge,
for example through the K75A mutation, could reduce telome-
rase activity. Taken together, these results highlight that a
direct association between Tpz1 and Trt1 promotes telomere
lengthening after telomerase has been recruited to telomeres.

B Ccq1 Is Required for Telomerase Activation after

Recruitment
Utilizing the Trt1-Tpz1 fusion system, we further asked
whether Ccq1 is also required for telomerase activity after
recruitment. First, to determine whether Est1 recruitment via
Ccq1 is still required for telomerase function when Trt1 is fused
to Tpz1, the T93A mutation was introduced into endogenous
ccq1+ in a trt1-tpz1 haploid strain. These cells displayed telo-
mere elongation comparable to strains expressing WT Ccq1
and the Trt1-Tpz1 fusion, indicating that fusion of Trt1 with
Tpz1 bypasses the need for Ccq1 in telomerase recruitment
(Figure 4C). Second, to investigate whether Ccq1 is required
for telomerase activation, the ccq1 gene was deleted in a
haploid strain expressing the Trt1-Tpz1 fusion protein. Strik-
ingly, telomeres were lost in ccq1D cells, indicating that
Ccq1 is required for telomerase activity after interaction of
Trt1 with Tpz1 (Figure 4C). Interestingly, whereas ccq1D single
mutants activate homologous recombination before complete
loss of the telomeric repeats [12], this was not observed in the
presence of the Trt1-Tpz1 fusion, suggesting that telomerase
C competes with the telomere-recombination pathway in the
absence of Ccq1. To investigate this further, we asked whether
it was necessary for Tpz1 within the fusion construct to
interact with Ccq1. A recent study described a single amino
acid mutation (L449A) within Tpz1 that disrupts the interaction
between Tpz1 and Ccq1 yet retains Ccq1 at telomeres [25].
Curiously, cells expressing the Trt1-Tpz1(L499A) fusion pro-
tein did not lose telomeres, although they were maintained at
a shorter length than in cells expressing the WT Trt1-Tpz1
fusion (Figure 4C). Collectively, these results indicate that
whereas fusion of Trt1 with Tpz1 bypasses the need for
Ccq1 in the recruitment process, Ccq1 is in fact required for
telomerase activity after recruitment.

Conclusions and Perspectives

Using protein fusions and loss-of-function mutations, we have
Figure 3. Tpz1 K75 Is Not Involved in Telomerase Recruitment been able to dissect the events of telomerase recruitment and
(A) Association efficiency of Trt1 with Tpz1 increases when K75 is mutated activity. The reduced telomerase activity observed in strains
to alanine. WCEs were immunoprecipitated with anti-HA antibody to purify containing the tpz1-K75A mutation despite accumulation of
Tpz1. The resulting immunoprecipitates were hybridized with anti-PK. Cdc2 Trt1 at the telomere demonstrates that association of telome-
was used as a control for sample input. rase with telomeres is not sufficient to regulate telomere
(B) Telomere ChIP: Trt1 is present at the telomere in strains carrying the
length. Furthermore, the interaction between Ccq1 and Est1
tpz1-K75A mutation. Strains were crosslinked, and WCEs were subjected
to immunoprecipitation with anti-PK antibody. The trt1-PK tpz1-K75A that is required for telomerase recruitment appears to be
cells were prepared soon after germination from the heterozygous transient, with a more stable association between Trt1 and
diploid. DNA fragments in the immunoprecipitate were quantified
using quantitative PCR (qPCR). Data were obtained from four indepen-
dent experiments, and normalized to qPCR values were obtained from (C) Association efficiency of Trt1 with Ccq1 increases when K75 of Tpz1 is
a control gene sequence (act1) and expressed as fold enrichment over mutated to alanine. The interaction of Ccq1 with Tpz1 is not affected by sub-
the values obtained from crosslinked WT (untagged) cells; the average stitution of Tpz1 K75 with alanine. WCEs were immunoprecipitated with
and SD of four independent experiments are shown. *p = 0.0289 for anti-Flag antibody to purify Ccq1. The resulting immunoprecipitates were
‘‘no-tag versus Trt1-PK.’’ *p = 0.0209 for ‘‘Trt1-PK versus Trt1-PK tpz1- hybridized with either anti-HA or anti-PK. Cdc2 was used as a control for
K75A.’’ sample input.
Current Biology Vol 24 No 17
2010

Tpz1-Ccq1 being achieved after recruitment. Specifically,

A B interaction of Trt1 with Tpz1 is crucial for telomere extension,
and Ccq1 is essential for telomerase activation. Collectively,
our data illustrate that telomerase recruitment and activation
are separate events (discussed further in Figure S4C) and
highlight the previously uncharacterized importance of Tpz1
and Ccq1 in regulating the latter. Such a two-step mechanism
may well be conserved from yeast to human.
In many human cancer cells, telomerase is highly expressed
and recruited to all telomeres, but processivity is low, resulting
in maintenance of short telomeres [28, 29]. Our findings shed
light on the existence of a telomerase regulatory step after
recruitment. Further investigation of telomerase activation
mechanisms would benefit our understanding of how cancer
cells maintain short telomeres that lead to chromosome
instability.

Supplemental Information

Supplemental Information includes Supplemental Experimental Proce-

dures, four figures, and one table and can be found with this article online
at http://dx.doi.org/10.1016/j.cub.2014.07.035.

Author Contributions

K.T. and C.A.A. were responsible for study design, experimental work, data
analysis, and manuscript preparation. S.R.P. carried out yeast two- and
three-hybrid assays. H.A. and V.M. contributed to immunoprecipitation as-
says and manuscript preparation.

C Acknowledgments

We thank Julia Promisel Cooper (NIH) for comments on our manuscript

before submission. This work is supported mainly by Cancer Research
UK (C36439/A12097) and partly by the European Research Council
(281722-HRMCB) and the Cancer Research UK UCL Centre.

Received: May 30, 2014

Revised: July 10, 2014
Accepted: July 14, 2014
Published: August 14, 2014

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