Tpz1TPP1 SUMOylation reveals evolutionary conservation of SUMO-dependent Stn1 telomere association

Elongation of the telomeric overhang by telomerase is counteracted by synthesis of the complementary strand by the CST complex, CTC1(Cdc13)/Stn1/Ten1. Interaction of budding yeast Stn1 with overhang-binding Cdc13 is increased by Cdc13 SUMOylation. Human and fission yeast CST instead interact with overhang-binding TPP1/POT1. We show that the fission yeast TPP1 ortholog, Tpz1, is SUMOylated. Tpz1 SUMOylation restricts telomere elongation and promotes Stn1/Ten1 telomere association, and a SUMO-Tpz1 fusion protein has increased affinity for Stn1. Our data suggest that SUMO inhibits telomerase through stimulation of Stn1/Ten1 action by Tpz1, highlighting the evolutionary conservation of the regulation of CST function by SUMOylation.

appropriate epitope tags linked by an 8xGlycine linker.
To obtain the tpz1-K242R (tpz1-Snm) allele, an NdeI-linearized plasmid (pAB1623) with a tpz1 fragment containing the mutated region and a ura+ marker was transformed into yeast. The resultant colonies were counter-selected on 5'-fluoroorotic acid (5'-FOA) to select for recombination at the Sp tpz1 locus. The presence of the K242R mutation was verified by PCR with DO1497/DO1826 (819 bp product) followed by restriction enzyme digest with HaeIII (429 + 390 bp in tpz1-Snm; uncut in WT).

Affinity-purification of SUMOylated Tpz1
Fission yeast cells were grown to a cell density of 1 x 10 7 cells/ml in 110 ml YNG medium lacking leucine and containing 15 mM thiamine. Of these, 100 and 5 ml were centrifuged for 5 min at 4°C at 3000 rpm, and the cell pellets were stored at -20°C as the uninduced fractions (for affinity-purification and WCE preparation, respectively). Cells from the remaining 5 ml cultures were harvested separately and the cell pellets were washed twice with sterile distilled water before resuspension in 110 ml fresh YNG medium lacking leucine with no thiamine. These cultures were then grown for 22 hrs to a cell density of 1 x 10 7 cells/ml to allow 6xhis-SUMO expression. After 22hrs the cells were harvested as done previously and stored at -20˚C, this time, as the induced fraction.
Ni-NTA affinity-purification was performed as described previously [1]. Harvested cells (from the cell pellets derived from the 100 ml cultures) were resuspended in 5 ml ice-cold distilled water followed by the addition of 0.8 ml of freshly prepared NaOH/BME (1.85 M NaOH, 7.5 mΜ βmercaptoethanol). After incubating the tubes on ice for 20 min, 0.8 ml of 55% TCA was added, and the tubes were incubated on ice for a further 20 min. Cells were then pelleted by centrifugation at 8000 rpm for 20 min at 4°C. Cell pellets were resuspended in 1 ml of Buffer A (6 M guanidine HCl, 100 mM sodium phosphate pH 8.0, 10 mM Tris-HCl pH 8) and rotated for 1 hour at room temperature. Supernatants were collected by centrifuging the tubes at 16000g for 10 min at 4°C.
Ni-NTA agarose beads (Qiagen) were prepared by washing it three times with Buffer A containing 0.05% Tween-20. 20 μl of washed beads were added to the supernatant along with 0.05% Tween-20 and 15 mM imidazole. The final mixtures were incubated at room temperature on a rotating wheel overnight. Then, the tubes were spun at 200 rpm for 15 sec and supernatants were discarded. The beads were washed twice with Buffer A plus 0.05% Tween-20, and washed four times with Buffer C (8 M urea, 100 mM sodium phosphate pH 6.3, 10 mM Tris-HCl pH 6.3) plus 0.05% Tween-20. 30 μl HU buffer (8 M urea, 200 mM Tris-HCl pH 6.8, 1 mM EDTA, 5% SDS, 0.1% bromophenol blue, 1.5% DTT) were added to the agarose beads. To prepare WCEs, pelleted cells were resuspended in 500 μl of distilled water followed by the addition of 75 μl of freshly prepared NaOH/BME and were incubated on ice for 15 min. 75 μl 55% TCA was added and the tubes were incubated on ice for a further 10 min. The lysates were centrifuged at 16000 rpm for 10 min at 4°C and the pellets were resuspended in 60 μl HU buffer. Both affinity-purified proteins and WCEs were heated at 60°C for 10 min before being analysed by SDS-PAGE.

Analysis of Tpz1 SUMOylation levels
Fission yeast strains containing the cdc25-22 allele were grown to mid-log-phase at 25°C, arrested in G2 for 3 hrs at 36°C and subsequently released at 25°C for 220 min. Samples were collected every 20 min after release and the septation index was determined. Cells were 3 resuspended in 600 μl 100%TCA, kept on ice for 10 min and then pelleted by centrifuging at 3000 rpm for 2 min, followed by two acetone washes. The pellets were then dried under vacuum and resuspended in 100 μl Urea buffer (50 mM Tris-Cl pH 7.5, 5 mM EDTA, 6 M Urea, 1% SDS). 200 μl 0.5 mm glass beads were added to the tubes and the cells were lysed in a bead beater 5 times for 45 sec with 1 min on ice in between. The extracts were incubated at 65°C for 10 min and centrifuged at 14000 rpm for 10 min before the addition of 200 μl of 2x Laemmli buffer. Samples were boiled for 5 min and separated on 8% SDS-PAGE gel. The proteins were visualized by ECL and imaged using a LAS 4000 instrument (GE).

Telomere length analysis by Southern blotting
Genomic DNA was prepared from log-phase cultures in YES rich media with NucleoSpin Sodium phosphate, pH7.2, 1% SDS) at 65°C for 5 min, followed by 20 min twice. Finally, the signals were visualized using a Fujifilm PhosphorImager Scanner (Molecular Dynamics).

Chromatin Immuno-precipitation
ChIP was performed as described previously [2,3]. 50 ml of log-phase cells were WCEs (10 µl) and IPs (40 µl) were taken to a final volume of 40 µl, 10 µl of denaturation buffer (1.5 M NaOH, 3 M NaCl) was added, and samples were incubated for 10 min at room temperature. 130 µl of dilution buffer (0.1x SSC, 0.125 M NaCl) were added and samples were incubated for 5 min on ice. The DNA was vacuum-spotted using a S&S Minifold I Dot Blot system onto a nylon membrane (Roche) pre-equilibrated with dilution buffer. The membrane was crosslinked, washed in neutralization buffer (0.5 M Tris-Cl pH 7.5, 0.5 M NaCl) and probed with a P 32 -labelled telomere probe (same as for Southerns). The signals were visualized using a Fujifilm PhosphorImager Scanner (Molecular Dynamics) and quantified using ImageQuant TL (GE Healthcare). In order to correct for overall differences in the efficiency of the immuno-precipitation between experiments, each experiment was normalized against its highest peak value.

Two-hybrid analysis
Yeast two hybrid assays were performed by co-transforming GBD (

Generation of the stn1-75 allele
A wild-type strain was transformed with a BstBI-HindIII fragment from pAB1447 to make a strain containing a loxP-Stn1-Ura4-loxM3 cassette integrated at the stn1 locus [4]. In this strain the cassette replaces the endogenous stn1 open reading frame with an identical one but devoid of introns. The intronless stn1 orf is under control of the endogenous stn1 promoter but is preceded by a loxP site. Strains bearing this cassette (stn1-LP allele) maintain telomeres, unlike a null stn1 allele, but these are of increased length, presumably due to altered expression levels. This strain was then transformed with BglII-linearized pAB1535 to delete rqh1+ in order to prevent circularization of chromosomes upon telomere loss [5], and subsequently with pAB1448, for thiamine-controlled expression of Cre recombinase. Error-prone PCR was used to amplify pAB1606 to generate PCR products containing a mutagenised loxP-Stn1-loxM3 cassette to be used for transformation of the stn1-LP strain. Transformants were plated on YNG-Leu plates without thiamine, incubated at 25°C for 2 days, before replica plating onto YES + 5-FOA and further incubated at 25°C. Once colonies were visible they were replica plated to two fresh 5-FOA plates, one of which was incubated at 25°C, the other at 36°C for another 2-3 days to identify temperature-sensitive alleles.