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. 2011 Nov 20;18(12):1408-13.
doi: 10.1038/nsmb.2187.

Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast

Bettina A Moser  1 , Ya-Ting Chang, Jorgena Kosti, Toru M Nakamura
Affiliations

Tel1ATM and Rad3ATR kinases promote Ccq1-Est1 interaction to maintain telomeres in fission yeast

Bettina A Moser et al. Nat Struct Mol Biol. .

Abstract

The evolutionarily conserved shelterin complex has been shown to play both positive and negative roles in telomerase regulation in mammals and fission yeast. Although shelterin prevents the checkpoint kinases ATM and ATR from fully activating DNA damage responses at telomeres in mammalian cells, those kinases also promote telomere maintenance. In fission yeast, cells lacking both Tel1 (ATM ortholog) and Rad3 (ATR ortholog) fail to recruit telomerase to telomeres and survive by circularizing chromosomes. However, the critical telomere substrate(s) of Tel1(ATM) and Rad3(ATR) was unknown. Here we show that phosphorylation of the shelterin subunit Ccq1 on Thr93, redundantly mediated by Tel1(ATM) and/or Rad3(ATR), is essential for telomerase association with telomeres. In addition, we show that the telomerase subunit Est1 interacts directly with the phosphorylated Thr93 of Ccq1 to ensure telomere maintenance. The shelterin subunits Taz1, Rap1 and Poz1 (previously established inhibitors of telomerase) were also found to negatively regulate Ccq1 phosphorylation. These findings establish Tel1(ATM)/Rad3(ATR)-dependent Ccq1 Thr93 phosphorylation as a critical regulator of telomere maintenance in fission yeast.

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Figures

Figure 1
Figure 1
Ccq1 interacts with both Tpz1 and Est1. (a) Schematic representation of Tpz1, Ccq1 and Est1. Conserved motifs and functional domains,,, are indicated. For Ccq1, putative consensus Tel1ATM/Rad3ATR phosphorylation sites (TQ or SQ) are indicated. For Est1, amino acid residues within the 14-3-3-like domain predicted to be important for phosphopeptide binding are marked. Gray shaded areas indicate regions required for protein-protein interactions, determined by yeast two-hybrid assays. (b-e) Determination of regions and amino acid residues critical for the Ccq1-Est1 interaction by yeast two-hybrid assays. Wild-type full-length proteins are denoted as “FL”. Truncation constructs are indicated as subscripts denoting amino acid residue numbers. Ccq1 mutants carrying multiple alanine mutations at SQ/TQ sites were abbreviated as 3AQ, 4AQ or 2AQ as indicated.
Figure 2
Figure 2
The phosphopeptide-binding motif of Est1 is important for telomere maintenance. (a) Southern blot analysis of telomeres from successive restreaks. While addition of a 13myc-tag to Est1 resulted in slightly shorter but stable telomeres, mutations within the 14-3-3-like domain caused loss of telomeres for both tagged and untagged mutant alleles (Supplementary Fig. 4). (b) NotI restriction map of fission yeast chromosomes. The telomeric fragments (C, I, L and M) are marked. (c) The telomeric NotI-fragments from extensively restreaked cells, analyzed by pulsed-field gel electrophoresis. (d) Binding of Est1 to TER1 is not affected by est1 mutations, as monitored by co-IP experiments. Plots show mean values plus/minus one s.d. for three independent experiments. (e-f) Recruitment of Est1 mutants (e) and Ccq1 in est1 mutant backgrounds (f) to telomeres was monitored by ChIP assays. Assays were performed using early generation cell cultures, and the presence of telomeres was confirmed by both Southern blot and qPCR assays against TAS regions, which are lost upon chromosome circularization, (data not shown). Protein expression levels were monitored by Western blot using the indicated antibodies. Cdc2 served as loading control. Plots show mean values plus/minus one s.d. for three (e) or two (f) independent experiments.
Figure 3
Figure 3
Ccq1 Thr93 is essential for telomere maintenance. (a) Removal of telomerase inhibitors (Poz1, Rap1 and Taz1) results in increased association of telomerase (Trt1TERT) with telomeres, as monitored by dot-blot ChIP assays (top panel). Plots show mean values plus/minus one s.d. for four independent experiments. Trt1 and Cdc2 expression levels were monitored by Western blot (bottom panel). (b-c) Western blot analysis of Ccq1. Asterisk (*) marks λ-phosphatase sensitive slow mobility band of Ccq1. Early generation tel1Δ rad3Δ strains were generated using the Rad3-plasmid loss system. (d) Southern blot analysis of telomeres during successive restreaks. (e) The telomeric NotI-fragments from extensively restreaked cells, analyzed by pulsed-field gel electrophoresis. (f) Quantitative real-time PCR ChIP analysis of Est1, Trt1 and Ccq1. ChIP assays of ccq1-T93A cells were performed using early generation cell cultures. Protein expression levels were monitored by Western blot. Plots show mean values plus/minus one s.d. for three (Est1 and Trt1) or two (Ccq1) independent experiments.
Figure 4
Figure 4
Ccq1–Est1 interaction is dependent on phosphorylated Ccq1 Thr93. (a) Binding of Ccq1 to telomerase RNA TER1 was monitored, and expression of Ccq1 was examined by anti-myc Western blot. Plots show mean values plus/minus one s.d. for at least three independent experiments. (b-c) Western blot of immuno-purified (anti-FLAG) Ccq1 using anti-phospho-(S/T)Q antibody (top panels), and of whole cell extracts (WCE) using anti-FLAG antibody (bottom panels). (d) Est1 specifically recognizes phosphorylated Thr93 of Ccq1. Extracts of wild-type Est1-myc strains were incubated with Streptavidin-beads bound to either T93 (non-phosphorylated), T93-P (phosphorylated), T93-P/Q94A or T93D peptides. For T93-P and T93-P/Q94A, peptides were also treated with λ-phosphatase (+) prior to incubation with extracts. Peptide bound Est1 was analyzed by Western blot analysis. (e) Est1 binding to phosphorylated Thr93 of Ccq1 is dependent on its 14-3-3-like phosphopeptide binding motif. Extracts of wild-type and 14-3-3-like domain mutant est1 strains were incubated with the T93-P peptide bound to Streptavidin-beads. (f) A model of telomere length regulation in fission yeast.
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