A Human Homolog of Yeast Est1 Associates with Telomerase and Uncaps Chromosome Ends When Overexpressed

Current Biology, Vol. 13, 568–574, April 1, 2003, 2003 Elsevier Science Ltd. All rights reserved. DOI 10.1016/S 09 60 - 98 22 ( 03 )0 0 17 3- 8

A Human Homolog of Yeast Est1 Associates with Telomerase and Uncaps Chromosome Ends When Overexpressed Patrick Reichenbach,1,3 Matthias Ho¨ss,1,3,4 Claus M. Azzalin,1,3 Markus Nabholz,1 Philipp Bucher,2 and Joachim Lingner1,* 1 Swiss Institute for Experimental Cancer Research (ISREC) 2 Swiss Institute for Bioinformatics Ch. des Boveresses 155 1066 Epalinges Switzerland

Summary Telomeres protect the eukaryotic chromosome ends from degradation and fusion. They are maintained by the ribonucleoprotein telomerase, the core of which is composed of a reverse transcriptase (TERT) and a RNA subunit. In the yeast Saccharomyces cerevisiae, a third critical telomerase subunit, the Ever Shorter Telomeres 1 (EST1) gene product, recruits or activates telomerase at the 3ⴕ end of telomeres [1–3]. Est1p has so far only been known in budding yeast, and mechanisms that mediate telomerase access and activation in other eukaryotes have remained elusive. Here, we use iterative profile searches to identify homologs of yeast Est1p in a large variety of eukaryotes, including human. One of three human homologs, designated human EST1A (hEST1A), is shown to be associated with most or all active telomerase in HeLa cell extracts. Overexpression of hEST1A induces anaphase bridges due to chromosome-end fusions, and telomeric DNA persists at the fusion points. Thus, overexpression of hEST1A affects telomere capping. The identification of EST1 homologs in a large variety of eukaryotes may indicate that the mechanisms of telomere extension are more conserved than anticipated previously. Results and Discussion Telomerase is the ribonucleoprotein enzyme that extends chromosome 3⬘ ends by iterative reverse transcription of the template region of its tightly associated RNA moiety [4]. While structural and functional features of the telomerase reverse transcriptase and the telomerase RNA moiety have been conserved throughout evolution [5–7], virtually no information is available on factors involved in telomerase recruitment and activation in species other than budding yeast. In 1989, a genetic screen in S. cerevisiae led to the discovery of the EST1 gene [8]. Deletion of this gene induces a progressive decrease in telomere length and a gradual loss of viability called replicative senescence. In the following years, genes of four other components of the telomere *Correspondence: [email protected] 3 These authors contributed equally to this work. 4 Present address: Inpharmatica, 2 Royal College Street, London NW1 0TU, United Kingdom.

replication machinery were identified (EST2, EST3, CDC13/EST4, and TLC1) that displayed the same Est⫺ phenotype when mutated [9, 10]. TLC1 and EST2 encode the telomerase RNA and the catalytic subunit of telomerase, respectively, and are the only two components known to be required for in vitro telomerase activity [11, 12]. EST4 affects the telomerase recruitment function of the multifunctional telomere binding protein Cdc13p [2, 13–15]. Biochemical characterization of Est1p revealed that this protein binds in vitro to the G-rich, single-stranded telomeric overhang and that this DNA binding requires a free 3⬘ end [16]. Est1p is physically associated with yeast telomerase, as shown in coimmunoprecipitation experiments with epitope-tagged Est1p [17–19], and it appears to bind directly to Tlc1 [20]. Est1p has also been shown to interact genetically and physically with Cdc13p [2, 15], the other important single-stranded telomeric DNA binding protein in yeast. The precise role of Est1 in telomere extension is controversial. A series of experiments with fusion proteins provided evidence for a model in which telomerase is recruited to the telomeric 3⬘ end by Est1p, which in turn interacts with telomere-bound Cdc13p [1, 2]. Based on chromatin immunoprecipitation analyses of associations between telomere end replication factors and the telomere through the cell cycle, an alternative “activation model” has been proposed [3]. According to this model, telomerase is constitutively present at the telomere in an inactive state. In late S phase, Est1p associates with the bound telomerase and, upon further interaction with Cdc13p, activates telomerase for synthesis. Est1p has so far only been known in budding yeast. We employed iterative profile searches to identify proteins with sequence similarity to yeast Est1p. First, a generalized profile [21] was constructed with the conserved N-terminal regions of the yeast Est1 and Ebs1 proteins, the latter being an Est1-related protein of unknown function [19]. This allowed identification of a putative homolog in S. pombe (SPBC2D10.13; also see the accompanying paper by Beernink et al. [22]) and refinement of the profile. Subsequent iterative profile searches identified additional Est1-related proteins in eukaryotes of distant phyla, including Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, and Homo sapiens (Figure 1). In human, there are three obvious homologs encoded by the cDNA clones KIAA0732, KIAA1089, and FLJ31473 (sequence accession numbers AB018275, AB029012, and AK0506035, respectively). Hereafter, the protein encoded by the KIAA0732-like cDNA clone will be referred to as hEST1A, as we were able to show that it functions in telomere regulation. The 5⬘ end of the hEST1A cDNA was cloned by 5⬘ RACE (see the Experimental Procedures in the Supplemental Data available with this article online). The proteins encoded by AB029012 and AK0506035 are referred to as hEST1B and hEST1C, respectively. Their

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Figure 1. Sequence Analysis of EST1-like Proteins (A) Domain architecture of EST1-related proteins: green, EST1 domain; blue: conserved region consisting of one and a half tetratricopeptide repeats (TPR); red, PilT amino-terminal (PIN) domain. The EST1 domains were identified by iterative profile searches with the PFTOOLS software package (see the Experimental Procedures in the Supplemental Data). The assignment of the TPR regions and the PIN domains is based on [24]. (B) Multiple sequence alignment of EST1 domains. The alignment was generated with ClustalW [42]. Conserved amino acids (http:// www.ch.embnet.org/software/ BOX_form.html) are indicated as white letters on a black background. Similar amino acids are emphasized with a gray background. It is notable that several EST1 domain-containing proteins (e.g., Sc Ebs1, SpSPBC2F12-03C, DM Anon34Ea, Dm CG6369) may not have a function at telomeres. (C) A phenogram showing the distance relationship of the EST1 domain sequences. The phylogenetic tree was calculated with ClustalW [42] from the alignment shown in Figure 1B.

potential functions at telomeres remain to be investigated. The yeast proteins Est1 and Ebs1 also share a conserved C-terminal domain, but iterative profile searches with this domain did not identify significant similarities to other proteins. However, with a profile made from conserved C-terminal motifs shared between hEST1A and Drosophila anon-34Ea, the latter being a protein of unknown function, we were able to detect a weak but significant hit to the bacterial protein PhoH. This protein appears to have evolved from the N-terminal part of a DNA or RNA helicase [23]. The results from our computational analysis of yeast Est1 homologs were recently complemented and partly confirmed [24]. Figure 1A presents a synthetic view of the previous and current studies. Clissold and Ponting found virtually the same set of proteins by an iterative profile search starting from a marginal sequence similarity between the N-terminal regions of Drosophila anon34Ea and C. elegans SMG-5. They presented statistical evidence that the sequence similarities found in the C-terminal region represent a PilT amino-terminal (PIN) domain region. A hallmark of PIN domain proteins are five conserved acidic (aspartate or glutamate) residues,

which are indicative of enzymes that ligate divalent cations. It was further suggested that PIN domain-containing proteins are related to 5⬘ nucleases [24]. This would be consistent with a hypothesis that the metazoan homologs of yeast EST1 use this region for binding to single-stranded nucleic acids. Whether or not 5⬘ nuclease activity has been retained remains to be tested. The previously published N-terminal sequence homologies comprise only the second half of the conserved N-terminal domain [24] identified here. Based on analysis of the C. elegans proteins SMG-5 and SMG-7 implicated in nonsense-mediated decay [25], Clissold and Ponting adopted the view that this region consists of one and a half tetratricopeptide repeats (TPR), proteinprotein interaction modules that occur in functionally different prokaryotic and eukaryotic proteins. To incorporate these results, we split our initially defined N-terminal domain into two modules, the first being a new domain named EST1 and the second being referred to as TPR (Figure 1A). An alignment of the EST1 domain sequences is shown in Figure 1B. A phylogenetic tree based on this alignment (Figure 1C) suggests that hEST1A is most closely related to C. elegans Y54F10AL-2. The human protein FLJ31473 (hEST1C) is also a member

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Figure 2. Coimmunoprecipitation of hEST1A and Telomerase Activity from HeLa Cells (A) Western blot analysis of total protein extracted from 293T cells transfected with an empty vector (left) or with a vector expressing GFP-hEST1A from the CMV promoter. The blot was probed with a ␣hEST1A antibody generated in rabbits with a synthetic 40 amino acid peptide from the C terminus of hEST1A. (B) Immunoprecipitation of recombinant GST-hEST1A protein expressed in E. coli with ␣hEST1A antibody and controls as indicated below the blot. The supernatant (SN) and bound fraction (B) were loaded. The blot was probed with ␣hEST1A antibody. (C) Telomerase was affinity purified from HeLa cell nuclear extracts approximately 200-fold by using an oligonucleotide that was complementary to the template region [26, 27]. Enriched telomerase was incubated with unspecific IgG (lanes 1–4), affinity-purified ␣hEST1A antibody (lanes 5–16), or antihTERT antibody (lanes 17–20). In the fractions corresponding to lanes 9–12, a 3.5-fold molar excess of the antigenic peptide was added, and, in lanes 13–16, a peptide of unrelated sequence was included with the antibody. Antibody-bound material was immobilized on Protein A-covered magnetic beads. Upon extensive washing, telomerase activity (dilutions of 1:5 and 1:10 for each pair of lanes) was determined in the supernatant and the bound fraction and was quantified on a PhosphoImager (indicated at the bottom).

of this subfamily, but it lacks the C-terminal PIN domain. Whether the EST1 domain and TPR region form independent modules is not clear. The fact that the former is always followed by the latter suggests that they form a single structural unit, in which ancient TPR repeats have been recycled to serve new functions. One of the hallmarks of yeast Est1 is its association with the telomerase complex. Therefore, we tested whether the human homolog is associated with active telomerase. A polyclonal antibody was raised in rabbits against a peptide corresponding to 40 amino acids of the hEST1A C terminus (see the Experimental Procedures in the Supplemental Data). The affinity-purified antibody was active in Western blots and specifically detected ectopically expressed hEST1A (Figure 2A), but not endogenous hEST1A, which may be expressed at very low levels. Consistent with low expression levels, the signal for hEST1A mRNA in Northern blots was very weak in different human tissues (see Figure S1 in the Supplemental Data). The ␣hEST1A antibody very efficiently immunoprecipitated recombinant hEST1A protein expressed in E. coli (Figure 2B). To test for the presence of the hEST1A epitope in the telomerase complex, immunoprecipitations were performed with a human telomerase fraction that had been affinity purified approximately 200-fold from HeLa cell nuclear extracts [26, 27]. Antibodies against hEST1A precipitated 69% of total telomerase activity (Figure 2C, lanes 5–8). Antibodies against the catalytic subunit of human telomerase

hTERT, which served as a positive control, precipitated 35% of the activity (lanes 17–20), whereas a nonspecific rabbit IgG failed to immunoprecipitate significant amounts of telomerase activity (lanes 1–4). Immunoprecipitation of telomerase activity by antibodies against hEST1A was completely abolished by a 3.5 M excess of the peptide against which the antibody was raised (lanes 9–12), whereas a nonspecific peptide did not compete (lanes 13–16). The peptide immunogen corresponds to a sequence of hEST1A that is not conserved in any of the other hEST1 homologs; this renders it highly unlikely that the antibody recognizes another of the human EST1 homologs. In addition, the peptide immunogen does not share significant sequence similarity with other human proteins present in the databases. Therefore, our results support the notion that most or all active telomerase complexes contain the antigenic peptideencoding hEST1A. Independently, Lea Harrington and colleagues [28] also identified hEST1A as a homolog of Est1p and present evidence for its association with telomerase. To explore the cellular function of hEST1A, we overexpressed the protein in the cervical cancer-derived HeLa cells, the fibrosarcoma-derived HT1080 cells, and telomerase-negative primary human lung fibroblasts (HLF) by transfecting with a plasmid containing the full-length hEST1A cDNA under the control of the strong CMV promoter. Transfection of the hEST1A-carrying plasmid induced a high rate of cell death in these three cell lines.

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The same toxic effect was observed when the hEST1A open reading frame was expressed in frame with various peptide tags (HA, VSV, tap) or with GFP. To investigate the fatal consequences of hEST1A overexpression, we used the HT1080-derived cell line HTC75 that expresses a tet-off repressor [29]. Cells were transfected with a plasmid containing tap-tagged [30] hEST1A under control of an inducible CMV promoter that contained seven tet operator sequences. Several clones were isolated by selection for plasmid-encoded puromycin resistance in the presence of doxycyclin, which enables the tet repressor to bind to the tet operator, and were screened by Western blot analysis for expression of transgenic hEST1A upon removal of doxycyclin (Figure 3A). Induction of hEST1A led to a rapid growth arrest, which was accompanied by dramatic morphological changes (Figure 3A). Some cells had a reduced size or displayed irregular extensions and fragmentation of their cytoplasm, whereas others had a vacuolated cytoplasm and contained multiple nuclei (Figure 3A). This was accompanied by the appearance of cells with a sub-G1 DNA content, as determined by flow cytometry (Figure 3B). Inspection of DAPI-stained nuclei uncovered an increased chromatin condensation, nuclear breakage, and DNA fragmentation (Figure 3C). Parallel experiments performed on cells transfected with the empty vector showed no effect on HTC75 cells. Time course analysis of DAPI-stained cells upon induction of hEST1A revealed the accumulation of anaphase bridges (Figure 3D). At day four, 57% of the anaphases from the induced cells showed chromosome bridges, while, in the uninduced sample, the frequency remained at about 20% (Figure 3D), which may be due to leakage of the inducible system. In a control clone that was transfected with the empty vector, the incidence of anaphase bridges was around 10%. Analysis of metaphase chromosomes demonstrated that the anaphase bridges correlated with the appearance of chromosomal end-to-end fusions (Figure 3E). Four days after induction of hEST1A, 46 metaphases from induced and uninduced samples were analyzed, and 69% and 30% of the metaphases, respectively, displayed at least one fusion event. FISH experiments with a fluorescently labeled PNA (CCCTAA)3 probe detected telomeric TTAGGG repeats at the sites of fusion (Figure 3F). No reduction of telomeric signals at fused chromosome ends was apparent when FISH was used (Figure 3F), and Southern blot experiments failed to reveal significant telomere shortening (see Figure S2 in the Supplemental Data). We conclude that overexpression of hEST1A disrupts the telomeric capping function without severely perturbing telomere length. The apoptotic-like phenotype may be the secondary consequence of the breakage of dicentric chromosomes, visible as anaphase bridges. Alternatively, the uncapped telomeres may directly induce an apoptotic response [31]. The uncapping of chromosome ends followed by endto-end chromosome fusions was previously observed upon overexpression of a dominant-negative allele of TRF2, which prevents binding of endogenous TRF2 to telomeres [29]. To test if overexpressed hEST1A perturbed the telomeric localization of TRF2, we transiently transfected HeLa cells with a plasmid expressing a GFP-

hEST1A fusion protein from the CMV promoter and monitored the localization of TRF2 by immunofluorescence microscopy. One day posttransfection, GFP-hEST1Aexpressing cells were detected by epifluorescence of GFP. Endogenous TRF2 was detected with a mouse antibody (Figure 4B). GFP-hEST1A is nuclear and enriched in the nucleolus (Figure 4A). TRF2, which gives a punctate staining due to its association with telomeres [29], was indistinguishable in cells that overexpressed GFP-hEST1A (see filled arrow in right panel of Figure 4B) and in cells that lacked transgene expression (empty arrow). This rules out the possibility of a substantial displacement of endogenous TRF2 from telomeres upon hEST1A overexpression. Hence, overexpressed hEST1A may mediate its effects by removing other telomere capping factors such as Pot1 [32], Ku [33–35], or DNA-PKcs [36–38] from telomeres. Alternatively, overexpressed hEST1A may modulate higher order DNA structures, such as t loops [39] or G-quartet structures, that have been implicated in telomere capping. The observed phenotypes provide compelling evidence for an involvement of hEST1A in telomere metabolism. Whether the uncapping activity of overexpressed hEST1A is physiologically relevant is unclear. It is conceivable that telomerase activity in vivo depends on factors that remodel the telomere cap structure, which may be inaccessible to telomerase due to coverage with telomere end binding proteins and/or due to the formation of higher order DNA structures such as t loops. An attractive hypothesis is that endogenous hEST1A enables in vivo telomerase activity by modulating the telomere structure. Recent evidence suggests that hEST1A is also involved in the nonsense-mediated mRNA decay pathway (NMD) [40]. Since NMD regulates expression levels of telomere components in yeast [41], it is also possible that overexpressed hEST1A affects telomere structure by controlling expression levels of telomere components via the NMD pathway. Biochemical experiments are required to elucidate the activities of hEST1A, and loss-of-function models must be established to test the requirement of hEST1A for telomerase function in vivo. The EST1 protein family members have in common a conserved N-terminal region that includes characteristic TPR repeats and a 100 amino acid region we refer to as the EST1 domain, whereas the C termini have diverged into several subfamilies. It is intriguing that the sequence similarity between EST1 homologs of S. cerevisiae [8], S. pombe [22], and the human EST1 (this study) does not extend to this region, while fission and budding yeast EST1 paralogs that are not required for telomere replication share sequence similarity over their entire lengths. The sequence similarity of EST1 proteins with SMG-5 and SMG-7, which are involved in nonsense-mediated mRNA decay and RNA interference, indicates that EST1-like proteins may play roles in diverse nucleic acid-dependent processes. Elucidation of the activities, functions, and domain organizations of telomerase-associated EST1 proteins in several eukaryotes will tell us whether the differences between them reflect an evolution in the structure and maintenance of the telomeres.

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Figure 3. Effects of hEST1A Overexpression on Cellular Growth, Genomic Stability, and Telomere Capping Images originate from a HTC75 clonal cell line overexpressing hEST1A (clone 3.6) and from an empty vector control clone (clone 1.1). (A) Upper panel: Western blot analysis for the expression of transgenic hEST1A of clone 3.6 in the presence (uninduced condition) and absence (induced condition) of doxycyclin after 1, 2, and 3 days as indicated. The blot was probed with rabbit IgG, which binds to the Protein A tag of the tap-tagged hEST1A. Middle panel: the growth curve upon expression of transgenic hEST1A. Relative cell numbers are indicated as a function of days in culture. Triangles represent clone 3.6; squares represent clone 1.1. Empty and filled symbols refer to cells grown in the presence (uninduced condition) and absence (induced condition) of doxycyclin, respectively. Lower panel: DIC optic analysis of the terminal phenotype of hEST1A-expressing clone 3.6 grown for 5 days in the presence (uninduced) or absence (induced) of doxycyclin. Small arrows point to shrunk or fragmented cells. The large arrow points to a large multinucleated cell. (B) FACS analysis upon induction of hEST1A. The cell number is indicated as a function of the relative DNA content based on staining with propidium iodide. Days of induction are indicated on the left. (C) DAPI staining of nuclei of clone 3.6. The right panel shows hEST1A expression induced for 5 days. (D) Anaphase bridges after overexpression of hEST1A. Upper panel: representative examples of anaphase bridges are shown in inverted DAPI staining. Lower panel: graph showing the time-dependent accumulation of anaphase bridges in clone 3.6 upon induction of hEST1A. Each time point corresponds to two independent experiments in which a total of 100 anaphases were scored. Numbers represent the percentage of anaphases with bridges as a function of days of hEST1A induction, and error bars refer to standard deviations between the two experiments. Light and dark blue represent empty vector control clone 1.1 grown in the presence and absence of doxycyclin, respectively. Light and dark green represent clone 3.6 grown in the presence (uninduced) or absence (induced) of doxycyclin, respectively.

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Figure 4. Effects of hEST1A Overexpression on Localization of TRF2 in HeLa Cells (A) GFP-hEST1A is nuclear and enriched in the nucleolus. From left to right: GFP-hEST1A is detected by epifluorescence; the nucleolar marker nucleolin is detected with a mouse monoclonal antibody; DNA is detected by DAPI. Unfused GFP is mostly nuclear, but not nucleolar, and to a lesser extent cytoplasmic (right). (B) GFP-hEST1A does not perturb localization of TRF2 at telomeres. Labeling is as in (A). The empty arrowheads point to nuclei that lack transgenic GFP-hEST1A, and the full arrowheads point to cells that express GFPhEST1A. A cell containing a fragmented nucleus is indicated by “D.” Cells were analyzed 1 day posttransfection.

Supplemental Data Supplemental Data including descriptions of the Experimental Procedures used and additional results in the form of two supplemental figures are available at http://images.cellpress.com/supmat/ supmatin.htm. Acknowledgments We thank J. Promisel Cooper for discussion and for sharing unpublished results and comments on the manuscript, L. Harrington for sharing unpublished results, A.-L. Ducrest for FACS analysis, and T. de Lange for HTC75 cells. This work was supported by the Human Frontiers Science Program and grants from the Swiss National Science Foundation. Received: September 27, 2002 Revised: December 3, 2002 Accepted: January 14, 2003 Published: April 1, 2003 References 1. Evans, S.K., and Lundblad, V. (1999). Est1 and Cdc13 as comediators of telomerase access. Science 286, 117–120. 2. Pennock, E., Buckley, K., and Lundblad, V. (2001). Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell 104, 387–396.

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(E) Analysis of metaphase chromosomes upon induction of hEST1A. Representative examples of fused chromosomes are on the right. Fusions (arrowheads) and centromeres (c) are indicated. (F) Telomeric TTAGGG repeats detected at the fusion points with a fluorescently labeled PNA (CCCTAA)3 probe (green). The same chromosomes are shown after FISH and after inverted DAPI staining. Labeling is as in Figure 3E.

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Accession Numbers The hEST1A cDNA sequence was deposited at GenBank with accession number AY145883.