Fission Yeast Shelterin Regulates DNA Polymerases and Rad3ATR Kinase to Limit Telomere Extension

Studies in fission yeast have previously identified evolutionarily conserved shelterin and Stn1-Ten1 complexes, and established Rad3ATR/Tel1ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 as the critical post-translational modification for telomerase recruitment to telomeres. Furthermore, shelterin subunits Poz1, Rap1 and Taz1 have been identified as negative regulators of Thr93 phosphorylation and telomerase recruitment. However, it remained unclear how telomere maintenance is dynamically regulated during the cell cycle. Thus, we investigated how loss of Poz1, Rap1 and Taz1 affects cell cycle regulation of Ccq1 Thr93 phosphorylation and telomere association of telomerase (Trt1TERT), DNA polymerases, Replication Protein A (RPA) complex, Rad3ATR-Rad26ATRIP checkpoint kinase complex, Tel1ATM kinase, shelterin subunits (Tpz1, Ccq1 and Poz1) and Stn1. We further investigated how telomere shortening, caused by trt1Δ or catalytically dead Trt1-D743A, affects cell cycle-regulated telomere association of telomerase and DNA polymerases. These analyses established that fission yeast shelterin maintains telomere length homeostasis by coordinating the differential arrival of leading (Polε) and lagging (Polα) strand DNA polymerases at telomeres to modulate Rad3ATR association, Ccq1 Thr93 phosphorylation and telomerase recruitment.


Figure S2
Analysis of Trt1 TERT recruitment to telomeres by dot blot-based asynchronous ChIP assays with telomeric DNA probe. (A) Telomere correction factors for Trt1-myc strains were established by determining telomere/rDNA hybridization signal ratios relative to wt cells. Telomere correction factors for other epitope tagged strains are shown in Supplementary Table S1 [25]. BrdU incorporation at telomeres is inhibited by addition of 15 mM HU for wt, poz1! and rap1! cells but not for taz1! cells. BrdU is incorporated into ars2004 with similar kinetics in the presence or absence of HU for all genetic backgrounds tested. (C) Pol1 (!) showed similar timing of recruitment to ars2004 in all genetic backgrounds tested. Error bars correspond to SEM.  TERT . Comparison of telomere length corrected ChIP data between Pol2 (") and Trt1 (A) or Pol1 (!) and Trt1 (B) in indicated genomic backgrounds. For explanation of shaded areas in graphs, see Figure 2 legend. Error bars correspond to SEM.

Figure S8
Raw % precipitated DNA against input DNA for Rad26 ATRIP (A), Rad3 ATR (B) and Rad11 RPA (C) obtained by dot blot-based asynchronous ChIP assays with telomeric DNA probe. Error bars correspond to SEM. (D) Anti-myc (Rad26 and Rad3) and anti-FLAG (Rad11) western blot analysis indicated comparable expression levels in different genetic backgrounds. Cdc2 western blot served as a loading control.

Figure S9
Raw % precipitated DNA against input DNA for Ccq1 (A), Tpz1 (B), Poz1 (C) and Stn1 (D) obtained by dot blot-based asynchronous ChIP assays with telomeric DNA probe. Error bars correspond to SEM. (E) Anti-myc western blot analyses indicated comparable expression levels for all proteins in different genetic backgrounds. Cdc2 western blot served as a loading control.

Figure S13
Comparison of peak normalized cell cycle ChIP data between Ccq1 and Tpz1. (A) Peak normalized ChIP data for either Ccq1 or Tpz1 in different genetic backgrounds were plotted to compare changes in temporal association with telomeres. (B) Comparison of peak normalized ChIP data indicated that temporal changes in telomere association for Ccq1 and Tpz1 are nearly identical in all genetic backgrounds tested. For explanation of shaded areas in graphs, see Figure 2 legend. Error bars correspond to SEM.

Figure S14
Comparison of peak normalized cell cycle ChIP data between Poz1 and Stn1. (A) Peak normalized ChIP data for either Poz1 or Stn1 in different genetic backgrounds were plotted to compare changes in temporal association with telomeres. (B) Comparison of peak normalized ChIP data indicated that temporal changes in telomere association for Poz1 and Stn1 are nearly identical in wt, rap1! and taz1! cells. For explanation of shaded areas in graphs, see Figure 2 legend. Error bars correspond to SEM.

Figure S16
Yeast 3-hybrid assay to monitor interaction between Tpz1 and Stn1-Ten1. Various truncation constructs of Tpz1 were tested for interaction with Stn1 and Ten1. Based on cell growth on -His selection plate, a Tpz1 fragment containing amino acids 224-420 was the smallest Tpz1 construct that retained interaction with Stn1 and Ten1. Based on growth on -His -Ade plate, a Tpz1 fragment containing amino acids 2-420 showed strongest interaction with Stn1 and Ten1.

Figure S19
Cell cycle ChIP assays for catalytically dead Trt1-D743A. (A) Telomere length analysis for indicated strains used in ChIP analysis. Genomic DNA was prepared from early generation strains. After digestion with EcoRI, DNA was fractionated on a 1% agarose gel and processed for Southern blot analysis with a telomere probe. (B) Raw % precipitated DNA against input DNA for Trt1 TERT obtained by real-time quantitative PCR analysis (left) or dot blot-based asynchronous ChIP assays with telomeric DNA probe (right). Trt1-D743A showed a statistically significant increase in telomere association compared to wt Trt1 TERT (p=5.4x10 -5 ) for PCR-based ChIP assay, independently confirming our conclusion from telomere-length corrected dot blot-based ChIP assay ( Figure 6B). Anti-myc western blot analysis indicated comparable expression levels of Trt1 in different genetic backgrounds. Cdc2 western blot served as a loading control.     ) were used to ensure that ChIP assays monitored trt1! or trt1-D743A cells carrying longest telomeres as possible.