Ssu72 phosphatase is a conserved telomere replication terminator
2019; Springer Nature; Volume: 38; Issue: 7 Linguagem: Inglês
10.15252/embj.2018100476
ISSN1460-2075
AutoresJosé Miguel Escandell, Edison Carvalho, María Gallo-Fernández, Clara C. Reis, Samah Matmati, Inês M. Luís, Isabel A. Abreu, Stéphane Coulon, Miguel Godinho Ferreira,
Tópico(s)DNA Repair Mechanisms
ResumoArticle22 February 2019Open Access Transparent process Ssu72 phosphatase is a conserved telomere replication terminator Jose Miguel Escandell Corresponding Author Jose Miguel Escandell [email protected] orcid.org/0000-0003-4857-9413 Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Edison SM Carvalho Edison SM Carvalho Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Maria Gallo-Fernandez Maria Gallo-Fernandez Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Clara C Reis Clara C Reis Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Samah Matmati Samah Matmati Equipe Labellisée Ligue, CRCM, CNRS, Inserm, Institut Paoli-Calmettes, Aix-Marseille University, Marseille, France Search for more papers by this author Inês Matias Luís Inês Matias Luís Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Isabel A Abreu Isabel A Abreu Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Stéphane Coulon Stéphane Coulon orcid.org/0000-0001-8090-914X Equipe Labellisée Ligue, CRCM, CNRS, Inserm, Institut Paoli-Calmettes, Aix-Marseille University, Marseille, France Search for more papers by this author Miguel Godinho Ferreira Corresponding Author Miguel Godinho Ferreira Miguel[email protected] orcid.org/0000-0002-8363-7183 Instituto Gulbenkian de Ciência, Oeiras, Portugal Institute for Research on Cancer and Aging of Nice (IRCAN), INSERM U1081 UMR7284, CNRS, Nice, France Search for more papers by this author Jose Miguel Escandell Corresponding Author Jose Miguel Escandell [email protected] orcid.org/0000-0003-4857-9413 Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Edison SM Carvalho Edison SM Carvalho Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Maria Gallo-Fernandez Maria Gallo-Fernandez Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Clara C Reis Clara C Reis Instituto Gulbenkian de Ciência, Oeiras, Portugal Search for more papers by this author Samah Matmati Samah Matmati Equipe Labellisée Ligue, CRCM, CNRS, Inserm, Institut Paoli-Calmettes, Aix-Marseille University, Marseille, France Search for more papers by this author Inês Matias Luís Inês Matias Luís Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Isabel A Abreu Isabel A Abreu Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal Search for more papers by this author Stéphane Coulon Stéphane Coulon orcid.org/0000-0001-8090-914X Equipe Labellisée Ligue, CRCM, CNRS, Inserm, Institut Paoli-Calmettes, Aix-Marseille University, Marseille, France Search for more papers by this author Miguel Godinho Ferreira Corresponding Author Miguel Godinho Ferreira [email protected] orcid.org/0000-0002-8363-7183 Instituto Gulbenkian de Ciência, Oeiras, Portugal Institute for Research on Cancer and Aging of Nice (IRCAN), INSERM U1081 UMR7284, CNRS, Nice, France Search for more papers by this author Author Information Jose Miguel Escandell *,1,‡, Edison SM Carvalho1,‡, Maria Gallo-Fernandez1, Clara C Reis1, Samah Matmati2, Inês Matias Luís3, Isabel A Abreu3, Stéphane Coulon2 and Miguel Godinho Ferreira *,1,4 1Instituto Gulbenkian de Ciência, Oeiras, Portugal 2Equipe Labellisée Ligue, CRCM, CNRS, Inserm, Institut Paoli-Calmettes, Aix-Marseille University, Marseille, France 3Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Oeiras, Portugal 4Institute for Research on Cancer and Aging of Nice (IRCAN), INSERM U1081 UMR7284, CNRS, Nice, France ‡These authors contributed equally to this work *Corresponding author. Tel: +351 214464511; E-nail: [email protected] *Corresponding author. Tel: +33 493377775; E-nail: [email protected] The EMBO Journal (2019)38:e100476https://doi.org/10.15252/embj.2018100476 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Telomeres, the protective ends of eukaryotic chromosomes, are replicated through concerted actions of conventional DNA polymerases and elongated by telomerase, but the regulation of this process is not fully understood. Telomere replication requires (Ctc1/Cdc13)-Stn1-Ten1, a telomeric ssDNA-binding complex homologous to RPA. Here, we show that the evolutionarily conserved phosphatase Ssu72 is responsible for terminating the cycle of telomere replication in fission yeast. Ssu72 controls the recruitment of Stn1 to telomeres by regulating Stn1 phosphorylation at Ser74, a residue located within its conserved OB-fold domain. Consequently, ssu72∆ mutants are defective in telomere replication and exhibit long 3′-ssDNA overhangs, indicative of defective lagging-strand DNA synthesis. We also show that hSSU72 regulates telomerase activation in human cells by controlling recruitment of hSTN1 to telomeres. These results reveal a previously unknown yet conserved role for the phosphatase SSU72, whereby this enzyme controls telomere homeostasis by activating lagging-strand DNA synthesis, thus terminating the cycle of telomere replication. Synopsis The conserved (CTC1)-Stn1-Ten1 complex regulates DNA lagging strand synthesis and telomerase activity. CDK1-dependent phosphorylation prevents Stn1 association with telomeres and is reversed by Ssu72 phosphatase, thereby allowing telomere overhang fill-in reaction and consequently telomerase inhibition. A genome-wide screen identifies Ssu72 as negative regulator of telomere elongation in fission yeast Ssu72 is required for semiconservative DNA replication by controlling Stn1-Pol α interaction Ssu72 regulates Stn1 phosphorylation in fission yeast at a novel and highly conserved phosphorylation site located at the OB fold domain. The role of Ssu72 phosphatase in controlling telomere elongation via Stn1 recruitment is conserved in human cells. Introduction Telomeres are protein-DNA complexes that form the ends of eukaryotic chromosomes (reviewed in Palm & de Lange, 2008). The predominant function of telomeres is to prevent the loss of genetic information and to inhibit DNA repair at chromosome ends, thus maintaining telomere protection and genome stability. Loss of telomere regulation has been linked to two main hallmarks of cancer: replicative immortality and genome instability (Hanahan & Weinberg, 2011). However, telomeres face an additional challenge: DNA replication. Due to G-rich repetitive DNA sequences and protective structures, telomeres represent a natural obstacle for passing replication forks (Maestroni et al, 2017), and replication fork collapse can lead to the loss of whole telomere tracts. To counteract these effects, telomerase (Trt1 in S. pombe and TERT in mammals) is responsible for adding specific repetitive sequences to telomeres, compensating for the cell's inability to fully replicate chromosome ends (Greider & Blackburn, 1985). However, it is currently incompletely understood how telomerase activity is regulated and how the telomerase cycle is coupled to telomeric DNA replication. Intriguingly, several DNA replication proteins are required for proper telomere extension (Dahlen et al, 2003). Conversely, specific telomere components are themselves required for proper telomere replication and telomere length regulation (Miller et al, 2006; Sfeir et al, 2009), suggesting that there is a thin line separating telomere replication and telomere elongation by telomerase. Using fission yeast, Chang et al (2013) proposed a dynamic model that demonstrates how telomere replication controls telomere length and how this is carried out by the telomere complex. The telomere dsDNA-binding components Taz1, Rap1, and Poz1 promote the recruitment of Polα-Primase to telomeres. Because shorter telomeres possess less Taz1/Rap1/Poz1, Polα-Primase recruitment and lagging-strand synthesis are delayed, leading to the accumulation of ssDNA at telomeres. This event results in the activation of the checkpoint kinase Rad3ATR and the subsequent phosphorylation of telomeric Ccq1-T93, a step required for telomerase activation in fission yeast. Thus, as a consequence of delayed Polα-Primase recruitment to short telomeres and the subsequent accumulation of ssDNA, Rad3ATR is transiently activated leading to telomerase recruitment and telomere elongation. Another complex known as CST (Cdc13/Stn1/Ten1 in S. cerevisiae and CTC1/STN1/TEN1 in mammals) is known to control telomere replication. This complex is responsible for both 5′-ssDNA strand protection from nucleolytic degradation and recruitment of the Polα-primase complex to telomeres, thus promoting telomere lagging-strand DNA synthesis (Lin & Zakian, 1996; Grossi et al, 2004). Notably, CST is not only required to recruit Polα-primase but is also responsible for the switch from primase to polymerase activity, which is required for gap-less DNA replication (Lue et al, 2014). In humans, in addition to its role in telomere replication (Surovtseva et al, 2009), the CST complex also functions as a telomerase activity terminator (Chen et al, 2012) by inhibiting telomerase activity through primer confiscation and direct interaction with the POT1-TPP1 dimer. However, the mechanism regulating these CST functions remains unknown. In fission yeast, although STN1 and TEN1 homologs exist, no Cdc13/CTC1 homolog has been identified to date (Martín et al, 2007). Recent studies revealed that Stn1 is required for replication of telomeres and subtelomeres (Takikawa et al, 2017; Matmati et al, 2018), supporting the conserved role of fission yeast (C)ST in DNA replication. In agreement with the replication model for telomere length regulation (Greider, 2016), the telomere-binding protein Rif1 was shown to regulate telomere DNA replication timing by recruiting Glc7 phosphatase to origins of replication and inhibiting Cdc7 activities in budding yeast (Hiraga et al, 2014; Mattarocci et al, 2014). Notably, this role is conserved in other organisms such as fission yeast (Davé et al, 2014) and human cells (Hiraga et al, 2017). Importantly, rif1+ mutants display long telomeres; this effect is suggested to be a result of origin firing dysregulation (Greider, 2016). However, how telomere replication is terminated and how this is coupled with the regulation of telomerase activity remain unknown. Here, we report that the protein phosphatase Ssu72 displays a conserved role as a telomere replication terminator. Ssu72 was previously identified as an RNA polymerase II C-terminal domain phosphatase and is highly conserved from yeast to human (Krishnamurthy et al, 2004). In addition, Ssu72 functions as a cohesin-binding factor involved in sister-chromatid cohesion by counteracting phosphorylation of the cohesion complex subunit SA2 (Kim et al, 2010). In fission yeast, in addition to regulating RNA polymerase activity, Ssu72 has been shown to regulate chromosome condensation (Vanoosthuyse et al, 2014). However, none of the previous studies have noted deregulated telomere replication. Our data strongly support an unexpected role for Ssu72 in controlling lagging-strand synthesis through the regulation of Stn1 serine-74 phosphorylation, thus reducing telomeric ssDNA and inhibiting telomerase recruitment. Results Ssu72 is a negative regulator of telomere elongation We carried out a genome-wide screen for regulators of telomere homeostasis in S. pombe using a commercially available whole-genome deletion library (Bioneer Corporation). This library allowed us to identify new non-essential genes involved in telomere homeostasis in fission yeast (Fig 1A). Of the genes identified from the screen, we selected the highly conserved phosphatase ssu72+ (SPAC3G9.04) as the most promising candidate for further characterization. We generated a deletion mutant (ssu72∆) as well as a point mutant devoid of phosphatase activity (ssu72-C13S) and found that these two mutants possess longer telomeres (Fig 1B). Additionally, we found that Ssu72 localized to telomeres in a cell cycle-dependent manner. We performed cell cycle synchronization using a cdc25-22 block-release method in a ssu72-myc-tagged strain and measured Ssu72 binding to telomeres by chromatin immunoprecipitation (ChIP). Cell cycle phases and synchronization efficiency were measured using the cell septation index. Interestingly, Ssu72-myc is recruited to telomeres in late S phase and declines later in the cell cycle (Fig 1C) and is recruited to telomeres at approximately the same time as the arrival of the lagging-strand machinery at chromosome ends (Chang et al, 2013). Figure 1. Genetic screen identifies Ssu72 as telomerase regulator We identified previously unknown telomere regulators in fission yeast using the haploid S. pombe whole-genome gene deletion library including ssu72+ (SPAC3G9.04). Telomere length of wt, ssu72Δ, and ssu72-C13S (point mutation at the phosphatase active site) strains were measured by Southern blot in ApaI-digested genomic DNA using a telomeric probe. Ssu72 recruitment to telomeres is cell cycle regulated. Ssu72 was myc-tagged at the 3′ end, and ChIP analysis was carried out in cell cycle synchronized populations of cdc25ts strains. Septa formation was used proxy for S phase. n ≥ 3; *P ≤ 0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM). Telomere length of ssu72Δ is dependent on telomerase. Diploid strains with the appropriate genotypes were sporulated, and trt1∆ ssu72∆ double mutants were streaked for multiple passages (triangle indicates increased number of generations). Telomerase is recruited to telomeres in the absence of Ssu72. ChIP analysis for Trt1-myc in wt and ssu72∆ was performed as described in Materials and Methods using a non-tagged strain as a control. n ≥ 3; *P ≤ 0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). Telomerase activator Ccq1 is hyperphosphorylated in ssu72∆ cells. rap1∆ cells were used as positive control for Ccq1 phosphorylation status. Western blots were performed using Ccq1-flag-tagged strains. Download figure Download PowerPoint ssu72∆ cells displayed increased (~1 Kb) telomere lengths compared to wild-type telomeres (~300 bp) (Fig 1B). We set out to understand the nature of telomere elongation in the ssu72∆ mutant background. To test whether the telomere elongation was dependent on telomerase, trt1∆ (deletion mutant for the catalytic subunit of telomerase) and ssu72∆ double heterozygous diploids were sporulated. Of the resulting tetrads, trt1∆ ssu72∆ double mutants were selected and streaked for several generations in order to facilitate telomere shortening in the absence of telomerase. While ssu72∆ mutant cells displayed long telomeres, ssu72∆ trt1∆ double mutant shortens telomeres (Fig 1D) in a passage-dependent manner. ChIP experiments consistently demonstrated an accumulation of Trt1-myc at ssu72∆ telomeres compared to wt cells (Fig 1E). Thus, the longer telomeres exhibited by ssu72∆ mutants were a consequence of telomerase deregulation. Two independent studies (Moser et al, 2012; Yamazaki et al, 2012) showed that Ccq1 phosphorylation at Thr93 is required for telomerase-mediated telomere elongation in fission yeast. Using Western blot shift analysis, we observed that Ccq1 was hyperphosphorylated in ssu72∆ cells when we compared to those of wt strains (Fig 1F). To further confirm that telomere elongation was telomerase-dependent, we repeated the previous experiment using a phosphorylation-resistant mutant version of Ccq1 (Moser et al, 2012). We germinated a double heterozygous ccq1-T93A/+ ssu72∆/+ mutant and analyzed its progeny. As expected, ccq1-T93A ssu72∆ double mutants displayed a similar telomere-shortening rate to that of the ccq1-T93A single mutants (Appendix Fig S1). In agreement with these results, we further showed that telomere length in ssu72∆ mutants was dependent on Rad3, the kinase responsible for Ccq1-T93 phosphorylation (Appendix Fig S2A), and not dependent on the checkpoint kinase Chk1 (Appendix Fig S2B). In addition, ssu72∆ rad51∆ double mutants displayed similar telomere lengths to ssu72∆ single mutants (Appendix Fig S2C). Taken together, our results demonstrate that Ssu72 is a negative regulator of telomerase, possibly counteracting Rad3 activation and Ccq1 phosphorylation. Telomere length regulation by Ssu72 phosphatase is synergistic with Rif1 In fission yeast, the presence of telomeric ssDNA results in Rad3 activation and telomere elongation (Moser et al, 2012). Thus, we investigated whether ssu72∆ mutants accumulated telomeric ssDNA. We carried out in-gel hybridization assays using a C-rich probe to measure the accumulation of G-rich DNA at telomeres. Notably, the ssu72∆ mutant strain showed an almost sixfold increase in G-rich telomere sequences (Fig 2A). We observed that the accumulation of ssDNA at telomeres is increased in ssu72∆ mutants compared to rif1∆ mutants, though both strains have similar telomere lengths. To control for the observed increase of ssu72∆ telomeric ssDNA resulted from chromosome termini, we treated both wt and ssu72∆ DNA with exonuclease I (Appendix Fig S3). As expected, exonuclease I treatment reduced the overhang signal in ssu72∆. In addition, we monitored Rad11RPA-GFP localization at telomeres in ssu72∆ mutant cells by live cell imaging and consistently detected an accumulation of this single-stranded binding protein at telomeres (Fig 2B). Thus, our data together suggest that ssu72∆ is defective in lagging-strand synthesis and possesses longer telomere overhangs. Figure 2. Ssu72 is required for telomeric C-strand stability ssu72Δ telomeres present longer G-rich overhangs than wt and rif1∆. In-gel hybridization in native and denaturing conditions was labeled with a radiolabeled C-rich telomere probe and quantified for ssDNA at the telomeres. n = 3; *P ≤ 0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). RPA (Rad11-GFP) is enriched at ssu72∆ telomeres. Co-localization of Rad11-GFP with Taz1-mCherry, used as a telomere marker, was performed in wt and ssu72∆ cells; n = 3; *P ≤ 0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). More than 1,000 cells were analyzed for each genotype ssu72+ controls telomere length independently of rif1+. Epistasis analysis of telomere length of ssu72∆ and ssu72-C13S (catalytically inactive mutant) with rif1∆ was performed by Southern blotting of ApaI-digested DNA using a telomeric probe. ssu72+ and stn1+ regulate telomere length in the same genetic pathway. Epistasis analysis of ssu72∆ and stn1-75 performed by Southern blotting of ApaI-digested DNA using a telomeric probe. Two independently generated ssu72∆ stn1-75 double mutants are shown. Download figure Download PowerPoint Recently, the telomere-binding protein Rif1 was found to control DNA resection and origin firing by recruiting PP1A phosphatase to double-strand breaks and origins of replication (Zimmermann et al, 2013; Davé et al, 2014; Hiraga et al, 2014; Mattarocci et al, 2014). We wondered whether Rif1 was also responsible for recruiting the Ssu72 phosphatase to telomeres. To test this hypothesis, we combined ssu72∆ and ssu72-C13S (catalytically dead) mutants with rif1∆ and carried out of telomere length epistasis analyses. While single mutants displayed telomere lengths of 1 Kb, both ssu72∆ rif1∆ and ssu72-C13S rif1∆ double mutants displayed telomeres that were longer than 3 Kb (Fig 2C). Thus, although Rif1 and Ssu72 do not control telomere length via the same genetic pathway and Rif1 is therefore not responsible for Ssu72 recruitment to telomeres, the observed synergistic telomere length effect provides evidence for crosstalk between the two regulatory mechanisms. Ssu72 controls telomere length through the Stn1-Ten1 complex CST constitutes a second highly conserved protein complex that regulates telomere length and telomerase activity. The budding yeast CST complex (Cdc13, Stn1, and Ten1) plays opposing roles at the telomeres. Cdc13 is required for telomerase recruitment and is activated through its interaction with Est1, a subunit of telomerase (Qi & Zakian, 2000). This interaction is promoted by the phosphorylation of Cdc13 at T308 by Cdk1(Cdc28). In contrast, the Siz1/2-mediated SUMOylation of Cdc13 at Lys908 promotes its interaction with Stn1 (Hang et al, 2011). This interaction is required, with Ten1, for polymerase alpha complex recruitment and telomere lagging-strand DNA synthesis (Grossi et al, 2004). However, the regulatory mechanism underlying these two opposite functions remains unknown. Despite the lack of Cdc13 homologs in fission yeast, the Stn1-Ten1 complex appears to play similar roles to those found in budding yeast and mammals (reviewed in Giraud-Panis et al, 2010). Consequently, we hypothesized that Ssu72 controls telomere length through the Stn1-Ten1 complex. Because fission yeast stn1+ and ten1+ deletion mutants lose telomeres completely and survive only with circular chromosomes (Martín et al, 2007), we carried out our experiments in mutants carrying a hypomorphic stn1-75 allele (Garg et al, 2014). Similar to ssu72∆ mutants, stn1-75 mutants possess long telomeres (~1 Kb) (Fig 2D). In contrast to our previous genetic studies presented in Fig 2C, stn1-75 ssu72∆ double mutants displayed similar telomere lengths to those of single mutants. This result suggests that Ssu72 controls telomere length through the same pathway as the Stn1-Ten1 complex. We then decided to investigate this genetic interaction using a different strategy. Fission yeast Stn1 is recruited to telomeres in a cell cycle-dependent manner (Moser et al, 2009a; Miyagawa et al, 2014), with peak telomere association in the S/G2 phases of the cell cycle. This coincides with Ssu72 recruitment to telomeres, as observed in our synchronization experiments (Fig 1C). Given the genetic association, we asked whether the recruitment of Stn1 to telomeres was dependent on the function of Ssu72. To test this hypothesis, we performed Stn1-myc ChIP experiments in ssu72∆ cells throughout the cell cycle (Fig 3A). As previously shown, Stn1-myc was recruited to telomeres in S/G2 cells (Moser & Nakamura, 2009). However, in the absence of Ssu72, the recruitment of Stn1 to telomeres was severely impaired (Fig 3A). Thus, our results show that Ssu72 function is required for cell cycle recruitment of Stn1 to telomeres. Figure 3. Ssu72 controls Stn1 telomere recruitment and phosphorylation Ssu72 is required for telomere recruitment of Stn1 in late S phase. ChIP analysis of stn1-myc in wt and ssu72∆ cells was performed in synchronized cdc25ts cells. n ≥ 3; *P ≤ 0.05 based on a two-tailed Student's t-test to ssu72+ control samples. Error bars represent standard error of the mean (SEM). 2D-gel analysis of NsiI telomeric fragments of wt and ssu72∆ strains. Smart ladder from Eurogentec was used for DNA size measurement. Serine-74 substitution to a phosphomimetic aspartate amino acid (stn1-S74D) is sufficient to confer ssu72∆ telomere defects. Telomere length epistasis analysis of ssu72∆ and stn1-S74D mutants was performed by Southern blotting of ApaI-digested genomic DNA using a telomeric probe. Sequence alignment of Stn1 highlighting serine-74 identified in fission yeast as a phosphorylated residue. Similar to ssu72∆ mutants, stn1-S74D is defective in telomere recruitment. ChIP analysis of stn1-myc and stn1-S74D-myc using a non-tagged strain as a control. n = 3; *P ≤ 0.05 based on a two-tailed Student's t-test to control sample. Error bars represent standard error of the mean (SEM). Download figure Download PowerPoint Based on these findings, we asked whether DNA replication was perturbed at chromosome ends of ssu72∆ mutants. Genomic DNA derived from wt and ssu72∆ cells was isolated, subjected to NsiI digestion and analyzed on 2D gels. Chromosome ends were revealed by Southern blotting using a telomere-proximal STE1 probe (Miller et al, 2006; Audry et al, 2015). In the first dimension, we observed three distinct bands for the wt parental strain but corresponding longer smearing bands for ssu72∆ mutants. As expected, we observed Y structures derived from passing replication forks within the NsiI fragment in wt cells (Fig 3B). In contrast, Y structures were not observed in ssu72∆ mutants denoting a reduced level of telomere DNA replication. In addition, DNA replication was 2–4 times lower at the rDNA locus in ssu72∆ mutants compared to wild-type cells (Fig 3B). These data are consistent with previous results for telomere and ribosomal DNA replication defects upon Stn1 inactivation (Takikawa et al, 2017), thus reinforcing the idea that Ssu72 controls Stn1 function throughout the genome. Ssu72 regulates Stn1 phosphorylation Given that Ssu72 phosphatase activity is required to regulate telomere length and that Ssu72 is recruited to telomeres during the S/G2 phases, we hypothesized that Ssu72 might regulate Stn1 phosphorylation in a cell cycle-dependent manner. Previous studies have revealed different Cdk1-dependent phosphorylation sites in Stn1 in budding yeast (Liu et al, 2014; Gopalakrishnan et al, 2017). However, to date, Stn1 phosphorylation sites have not been identified in species outside of S. cerevisiae. Moreover, the phosphorylation sites described for budding yeast are not conserved in S. pombe. Thus, we decided to take an unbiased approach using mass spectrometry-based analysis of purified fission yeast Stn1. We immunoprecipitated Stn1-myc from cells carrying the ssu72∆ deletion. Subsequent analysis of the immunoprecipitated material revealed a phosphorylated peptide corresponding to Stn1 serine-74 (Fig EV1A). Notably, this serine is not only conserved in Schizosaccharomyces sp (the fission yeast genus) (Fig 3B) and budding yeast but also throughout higher eukaryotes, including humans and mice (Fig 3D). Therefore, we decided to mutate the serine-74 residue to aspartic acid (Stn1-S74D), a phosphomimetic amino acid. Click here to expand this figure. Figure EV1. Identification of S74 as Stn1 phosphorylation site in fission yeast Mass spectrometry spectra identifying the phosphorylation on Stn1-S74 residue. S74 residue is conserved within the Schizosaccharomyces genus. Sequence alignment of Stn1 protein in Schizosaccharomyces genus (S. pombe, S. cryophilus, S. octosporus, and S. japonicus) using Clustal Omega. Serine-74 is colored in red. The site is either conserved or exchanged by an amino acid capable of being phosphorylated. Download figure Download PowerPoint Cells harboring the stn1-S74D mutation exhibited long telomeres (~1 Kb) similar to those found in ssu72∆ cells (Fig 3C). We hypothesized that telomere elongation in the stn1-S74D strain was telomerase-dependent. Consistent with this hypothesis, stn1-S74D trt1∆ double-mutant telomeres become shorter after sequential streaks (Fig EV2). Importantly, stn1-S74D ssu72∆ double mutants displayed similar telomere lengths to those in stn1-S74D single mutants (Fig 3C). In addition, we performed ChIP experiments in strains expressing Stn1-S74D-myc in order to analyze the recruitment of Stn1 to telomeres. Similar to our observations in mutants lacking ssu72 phosphatase, Stn1-S74D-myc was not efficiently recruited to telomeres (Fig 3E). Taken together, our data suggest that fission yeast Stn1 is dephosphorylated at Ser74 to enable its efficient recruitment to telomeres and, consequently, efficient DNA replication and telomerase regulation. Click here to expand this figure. Figure EV2. Telomere length of stn1-S74D is dependent on telomerasetrt1+ was deleted in the stn1-S74D background, and double mutants were streaked for multiple passages (triangle indicates increased number of generations). Download figure Download PowerPoint Given that both Stn1 and Ssu72 are recruited to telomeres in S/G2 phases of the cell cycle and that Stn1-S74 dephosphorylation is required for efficient Stn1 recruitment to telomeres, we wondered whether Ssu72 phosphatase could counteract the action of a cell cycle-dependent kinase. Due to the central nature of Cdc2Cdk1 in regulating the cell cycle, we mutated ssu72+ in cells carrying the cdc2-M68 temperature-sensitive mutant allele. At permissive temperatures (25°C), ssu72∆ cdc2-M68 cells exhibited a similar telomere length to ssu72∆ single mutants (Fig EV3A). To inactivate Cdc2 activity, we grew ssu72∆ cdc2-M68 strains at semi-permissive temperatures. Our results show that progressive inactivation of Cdc2Cdk1 in ssu72∆ cdc2-M68 double mutants resulted in a decrease in telomere length at 32 and 34°C when compared to 19 and 25°C and with ssu72∆ single mutants. In order to have a more direct readout for the involvement of Cdc2Cdk1 on Stn1 regulation, we sought to test whether we could rescue Stn1 recruitment defects in ssu72∆ background by inactivating Cdc2Cdk1 kinase activity. We took advantage of the recently published cdc2-as-M17 allele (Aoi et al, 2014) that is sensitive to ATP analogs and is optimized to eliminate other physiological limitations. We constructed ssu72∆ stn1-myc c
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