Research ArticleMOLECULAR BIOLOGY

The fission yeast Stn1-Ten1 complex limits telomerase activity via its SUMO-interacting motif and promotes telomeres replication

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Science Advances  16 May 2018:
Vol. 4, no. 5, eaar2740
DOI: 10.1126/sciadv.aar2740
  • Fig. 1 Mutations of the Stn1 SIM domain lead to elongated telomeres.

    (A and C) Y2H analysis of budding yeast strain carrying the indicated plasmids. The indicated proteins were fused either to B42 activating domain (B42-AD) or to LexA DNA binding domain. Cells were grown and spotted on selective medium (-TRP and -HIS) to select the two Y2H plasmids and tested for the activation of either Leu2 or LacZ reporters. (B) Schematic representation of the Stn1 and Ten1 proteins. The NStn1 contains the OB-fold domain that interacts with Ten1, whereas the CStn1 contains the WH1-2. The SIM domain that starts at position 226 is embedded into the WH1. The IRQM and the IIYL motifs mutated in the stn1-1 and stn1-195 alleles are also represented, respectively. (D) Analysis of viability of the indicated strains at different temperatures. Left: Cells were grown at 25°C, then serially diluted (fivefold), and plated on YES-rich medium either complemented or not with phloxine. Pink color reflects impaired growth. The stn1-226 allele carries mutations in SIM (226ILAL229 to 226AAAA229). Right: Growth curve of WT and stn1-226 strains. Both strains were cultured at 25°C in YES medium and then shifted to 36°C for 24 hours. (E) Genomic DNA from cells with the indicated genotype was prepared, digested with Apa I, and analyzed by Southern blotting with a radiolabeled telomeric DNA probe. (F) Stn1/Ten1 interaction is analyzed by coimmunoprecipitation (co-IP). Pulldown was carried out using anti-Flag (M2, mouse monoclonal antibody) from cell extracts prepared from indicated cultures carried out at 25°C if not specified. The co-IP of Stn1 was monitored using anti-myc (9E10, mouse monoclonal antibody). (G) Ten1-Stn1 interaction analyzed by Y2H. Cells were grown and spotted on selective medium (-TRP and -HIS) and tested for activation of LacZ reporter. Image is displayed on black and white background to visualize the blue color. (H) Ten1Stn1 interactions with SUMO, SUMO-Tpz1, and Tpz1 analyzed by Y2H. Cells were grown and spotted on selective medium (-TRP and -HIS) and tested for activation of Leu2 and/or LacZ reporters.

  • Fig. 2 Stn1 SIM mutations affect Stn1-Ten1 recruitment and enhance telomerase binding to telomeres.

    (A) Telomere association of Stn1, Ten1, and Est1 monitored with dot blot ChIP assays. ChIP experiments were performed using samples collected from 25°C exponentially growing WT stn1+ and stn1-226 cells carrying the indicated epitope-tagged proteins. Error bars indicate the SEM from multiple independent experiments (n = 3); P values are calculated from two-tailed t test (**P < 0.01 and ***P < 0.005). (B) Top: Growth of spores carrying the indicated genotypes. Bottom: Telomere length analysis of two tetrads by Southern blotting with a radiolabeled telomeric DNA probe. Genomic DNA was digested with Apa I restriction enzyme. (C) Left: PFGE stained by ethidium bromide (EtBr) of intact chromosomes from WT strain, parental tpz1K242R and stn1-226 single mutants, and tpz1K242R stn1-226 double mutants grown at 25°C. Chromosomes I, II, and III were stable in single mutants, whereas they did not enter into the gel in double mutants. Right: Not I–digested genomic DNA was subjected to PFGE, transferred to nitrocellulose membrane, and hybridized with probes of telomere proximal fragments C, I, M, and L. In tpz1K242R stn1-226 double mutants, these fragments were absent and have been replaced by DNA fragments C + M and L + I. This modification in the Not I–digested pattern is characteristic of chromosome circularization. (D) Ten1Stn1 interactions with Tpz1 analyzed by Y2H in different mutants. Cells were grown and spotted on selective medium (-TRP and -HIS) and tested for activation of Leu2 and/or LacZ reporters. Y2H interaction was quantified by measurement of β-galactosidase activity.

  • Fig. 3 Telomeric and subtelomeric regions are lost in stn1-226 at a high temperature.

    (A and B) WT and stn1-226 strains were cultivated at 25°C in YES medium and then shifted at 36°C for 24 hours. Genomic DNA was prepared, digested with Apa I restriction enzyme, and analyzed by Southern blotting with radiolabeled probes, telomeric probe (A), subtelomeric STE1 probe (B), and a chromosomic probe (used as a loading control). Quantification of normalized telomeric signal over chromosomic signal is shown in the lower panel. Error bars indicate the average from two independent experiments. At a restrictive temperature, both telomeric and subtelomeric signals disappeared in stn1-226. (C) Synthetic sickness of rad51Δ and stn1-226 alleles. Spore colonies, resulting from tetrad dissection of a mating between rad51Δ and stn1-226 mutants, were grown at 25°C. (D) Telomere length analysis of indicated strains grown at 25°C. Genomic DNA was prepared, digested with Apa I restriction enzyme, and analyzed by Southern blotting with radiolabeled telomeric, subtelomeric (STE1), and chromosomic probes (loading control). At a permissive temperature, the telomeric and subtelomeric signal disappeared in both rad51Δ stn1-226 double mutants. (E) Left: PFGE stained by ethidium bromide of intact chromosomes from WT strain, stn1-226, rad51Δ, and stn1-226 rad51Δ mutants grown at 25°C. Chromosomes I, II, and III entered into the gel. Right: Not I–digested genomic DNA was subjected to PFGE, transferred to nitrocellulose membrane, and hybridized with probes of telomere proximal fragments C, I, M, and L. The rad51Δ stn1-226 4D clone exhibited a partial circularization of its chromosomes, whereas clone 1D displayed a rearranged profile with partial circularization. (F) Analysis of viability of the indicated strains at 25° and 36°C. Cells were grown at 25°C, then serially diluted, and plated on YES-rich medium either complemented or not with phloxine.

  • Fig. 4 Stn1 is required to promote efficient replication of telomeric regions.

    (A) Relative position of the restriction sites in the subtelomeric regions of chromosomes I and II. The subtelomeric probe (STE1) that is used for 2D-gel hybridization (see below) is represented. Cen, centromere. Southern blot analysis of Nsi I telomeric fragments (first dimension) from the parental WT strain and stn1-226 mutant revealed by STE1 probe. (B) 2D-gel analysis of Nsi I telomeric fragments of WT and stn1-226 strains at 25°C and after 24 hours at 36°C. The first dimension of the EtBr-stained gel was photographed before the second dimension. The Y-arc pattern is generated by unidirectional movement of a replication fork across each telomeric fragment shown in the first dimension. The cone-shaped signal represents four-way DNA junctions (double Y). Quantification of the total replication intermediate signal over the linear arc signal of rDNA is presented. N.A., not available. (C) Top: Map of the rDNA repeats. Boxes indicate the ars3001 and the RFB pause sites (P). The restriction enzyme sites are indicated (H, Hind III; B, Bam HI; K, Kpn I; S, Sac I; E, Eco RI). Left: Diagram of the migration pattern of replication intermediates that can be detected by 2D-gel electrophoresis. Right: 2D-gel analysis of rDNA RFB site in WT and stn1-226 strains at permissive and restrictive temperatures. The first dimension of the EtBr-stained gel was photographed before the second dimension. Quantification of the total replication intermediate signal over the linear arc signal is presented. The Eco RI–Eco RI fragment is used as a probe.

  • Fig. 5 Genetic interactions of stn1-226 allele with rpa1-D223Y, taz1Δ, rif1Δ, rif1-PP1, and rap1Δ.

    (A to E) Tetrad dissections, telomere length analysis, and viability of the indicated spore colonies are shown. Analysis of viability was carried out at 25° and 36°C. Cells were grown at 25°C, then serially diluted (fivefold), and plated on YES-rich medium. Cells were grown at 25°C, and genomic DNA was digested with Apa I restriction enzyme for telomere Southern blotting analysis. Loading control corresponds to a 2.4-kbp fragment of a chromosomic region. (C) Left: PFGE stained by ethidium bromide of intact chromosomes from WT strain, parental taz1Δ and stn1-226 single mutants, and taz1Δ stn1-226 double mutants. Chromosomes I, II, and III were stable in single mutants, whereas they did not enter into the gel in double mutants. Right: Not I–digested genomic DNA was subjected to PFGE, transferred to nitrocellulose membrane, and hybridized with probes of telomere proximal fragments C, I, M, and L. In taz1Δ stn1-226 double mutant, these fragments were absent, suggesting that chromosomes are circular.

  • Fig. 6 Stn1-Ten1 promotes DNA synthesis at telomeres to limit ssDNA accumulation.

    (A) WT and stn1-226 cells were grown at 25°C, then serially diluted (fivefold), and plated on YES-rich medium in the presence of the indicated drugs. (B) WT and stn1-226 cells containing pREP41 (empty vector) or pREP41-Pol1 plasmids were grown at 25°C in selective medium (PMG UAH), then serially diluted (fivefold), and plated. Plates were incubated at 25°, 32°, and 36°C. (C) Plating efficiencies were measured for WT and stn1-226 cells containing pREP41 (empty vector) or pREP41-Pol1 plasmids. Cells were grown at 25°C in selective medium and enumerated, and approximately a thousand cells were plated in triplicate. Plates were incubated at 25°, 32°, and 36°C. The plating efficiency was calculated by determining the ratio of CFU at 32° and 36°C above CFU at 25°C. Error bars indicate the SEM from multiple independent experiments (n = 4); P values are calculated from two-tailed t test (****P < 0.0001). (D) Overexpression of Pol1 partially complements stn1-226 defects. stn1-226 strains containing either pREP41 (empty vector) or pREP41-Pol1 were cultivated at 25°C in selective medium (PMG UAH) and then shifted at 36°C for 24 hours. Genomic DNA was prepared, digested with Eco RI restriction enzyme, and analyzed by Southern blotting with radiolabeled telomeric and chromosomic probes. Quantification of telomeric signal over chromosomic signal is presented. Error bars indicate the SEM from multiple independent experiments (n = 3); P values are calculated from two-tailed t test (***P < 0.005). (E) Analysis of Nsi I–digested genomic DNA was performed using in-gel nondenaturing hybridization, using a telomeric C probe. The same gel was denatured and rehybridized with the same probe and then hybridized again with a chromosomic probe (loading control). The G-strand signal intensities were quantified, and the ratio of intensities for nondenatured signal over denatured loading signal was calculated and plotted. (F) Dual action of Stn1-Ten1 complex at telomeres. Left: The Stn1-Ten1 complex acts as a modulator of telomerase action through the SIM domain of Stn1 and likely promotes the synthesis of the complementary strand by facilitating Polα action at telomere extremities. Right: At the subtelomeric and telomeric regions, the slowdown of the replication fork may accentuate the accumulation of nascent ssDNA. Thus, accessory factors are required to promote the replications of telomeres. The Stn1-Ten1 complex is likely required to either initiate or facilitate de novo DNA synthesis by the Prim-Polα complex. The interaction of Stn1 with a sumoylated partner through its SIM domain remains hypothetical. Stn1-Ten1 acts in a pathway that is independent of Taz1 and also distinct from Rad51, which might be required to protect nascent ssDNA at stalled fork at telomeres.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/4/5/eaar2740/DC1

    table S1. Fission yeast strains used in this study.

    table S2. Plasmids used in this study.

    fig. S1. Comparison of Stn1-226 with other Stn1 mutants.

    fig. S2. Deletion of rif1+ and exo1+ restores viability of stn1-226 allele.

  • Supplementary Materials

    This PDF file includes:

    • table S1. Fission yeast strains used in this study.
    • table S2. Plasmids used in this study.
    • fig. S1. Comparison of Stn1-226 with other Stn1 mutants.
    • fig. S2. Deletion of rif1+ and exo1+ restores viability of stn1-226 allele.

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