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. 2009 Aug;29(16):4584-94.
doi: 10.1128/MCB.01879-08. Epub 2009 Jun 22.

TOR complex 2 controls gene silencing, telomere length maintenance, and survival under DNA-damaging conditions

Miriam Schonbrun  1 , Dana Laor, Luis López-Maury, Jürg Bähler, Martin Kupiec, Ronit Weisman
Affiliations

TOR complex 2 controls gene silencing, telomere length maintenance, and survival under DNA-damaging conditions

Miriam Schonbrun et al. Mol Cell Biol. 2009 Aug.

Abstract

The Target Of Rapamycin (TOR) kinase belongs to the highly conserved eukaryotic family of phosphatidylinositol-3-kinase-related kinases (PIKKs). TOR proteins are found at the core of two distinct evolutionarily conserved complexes, TORC1 and TORC2. Disruption of TORC1 or TORC2 results in characteristically dissimilar phenotypes. TORC1 is a major cell growth regulator, while the cellular roles of TORC2 are not well understood. In the fission yeast Schizosaccharomyces pombe, Tor1 is a component of the TORC2 complex, which is particularly required during starvation and various stress conditions. Our genome-wide gene expression analysis of Deltator1 mutants indicates an extensive similarity with chromatin structure mutants. Consistently, TORC2 regulates several chromatin-mediated functions, including gene silencing, telomere length maintenance, and tolerance to DNA damage. These novel cellular roles of TORC2 are rapamycin insensitive. Cells lacking Tor1 are highly sensitive to the DNA-damaging drugs hydroxyurea (HU) and methyl methanesulfonate, similar to mutants of the checkpoint kinase Rad3 (ATR). Unlike Rad3, Tor1 is not required for the cell cycle arrest in the presence of damaged DNA. Instead, Tor1 becomes essential for dephosphorylation and reactivation of the cyclin-dependent kinase Cdc2, thus allowing reentry into mitosis following recovery from DNA replication arrest. Taken together, our data highlight critical roles for TORC2 in chromatin metabolism and in promoting mitotic entry, most notably after recovery from DNA-damaging conditions. These data place TOR proteins in line with other PIKK members, such as ATM and ATR, as guardians of genome stability.

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Figures

FIG. 1.
FIG. 1.
Tor1 is required for gene silencing and maintenance of telomere length. (A) The set of genes upregulated by loss of Tor1 significantly overlaps with the set of genes upregulated in histone deacetylase mutants. The number of genes that were upregulated 1.5-fold in the indicated mutants is presented in Venn diagrams, along with corresponding P values. (B) Northern blot analysis. Total RNA was prepared from wild-type (WT), Δtor1, and clr6-1 mutants grown to mid-log phase in rich medium. Northern blots were probed with the indicated genes. (C) Tor1 promotes silencing at the mating type locus. Strains containing an ade6+ cassette at the mating type locus were spotted onto the indicated plates. In an otherwise wild-type background, the ade6+ gene insertion produced a typical position variegation effect, since only a portion of the colonies is white (express the ade6+ gene) while others are red due to a decreased level of ade6+ transcript and accumulation of a red pigment. Only white colonies are present in cells carrying the Δtor1 mutation. (D) Tor1 is required for tolerance to microtubule destabilizing agents. Cells were streaked on plates containing the indicated levels of TBZ. (E) Tor1 is required for the maintenance of telomere length regulation. DNA was extracted from cells grown in rich medium (or minimal medium; asterisk). When rapamycin was added (R), the cells were grown in the presence of 100 ng/ml rapamycin. Genomic DNA was digested with EcoRI, which in wild-type cuts about 1 kb from the terminus, and analyzed by Southern blotting. The resulting filter was probed with α32-P-labeled telomere repeat DNA.
FIG. 2.
FIG. 2.
Mutations in TORC2 but not TORC1 confer sensitivity to DNA replication stress in a rapamycin-independent manner. (A and B) TORC2 but not TORC1 components are required for HU tolerance. Strains were streaked on plates with or without the indicated amounts of HU. (C) Overexpression of gad8+ partially rescues the lethal phenotype of Δtor1 cells on HU. gad8+ is expressed from the thiamine (T)-repressible nmt1+ promoter from the plasmids pREP1, -41, and -81, which allow strong, moderate, and weak expression, respectively. tor1+ is expressed from a plasmid under the regulation of its own promoter. (D) Rapamycin does not affect tolerance to DNA-damaging conditions. Serial dilutions of wild-type cells in the presence of 2.5 mM HU or 0.0025% MMS or UV irradiated at 75 J/m2 with or without 100 ng/ml rapamycin (R).
FIG. 3.
FIG. 3.
Mutations in TORC2 confer sensitivity to DNA-damaging conditions independently of Cds1 or Chk1. (A and B) Tor1 functions independently of Chk1 or Cds1. Serial dilutions of mutant cells were plated with or without the indicated amounts of HU or MMS. (C) Tor1 is not required for phosphorylation of Chk1. Western blot analysis of HA-tagged Chk1. Wild-type or Δtor1 cells containing HA-tagged Chk1 were grown to log phase. Protein was extracted from untreated cells or treated with 0.2% MMS for the indicated times (minutes).
FIG. 4.
FIG. 4.
Tor1 is required for a normal response to HU. (A and B) The response of tor1 mutants to HU is delayed. Wild-type and Δtor1 cells were grown to log phase and shifted to medium containing 12 mM HU. (A) Samples were taken every hour, and nuclei were isolated and subjected to FACS analysis. (B) Total RNA was prepared from samples taken at the indicated time points (hours) after a shift to 12 mM HU. Northern blots were probed with cdt2+ and cdc18+ (MBF targets) and with act1+ (loading control). (C) Loss of Tor1 rescues the mitotic catastrophe of Δrad3 mutants. Cells were incubated with or without 12 mM HU for 6 h at 30°C and then stained with DAPI and calcofluor to visualize nuclear DNA and septa, respectively. Percentages indicate abnormal mitosis, scoring for the “cut” phenotype in which the septum is formed despite the absence of chromosome replication. (D) The rapid loss of viability of the Δrad3 or Δcds1 mutant strain is rescued by Δtor1. Cells were grown to log phase and shifted to 12 mM HU for 6 h, and samples were taken every hour to determine cell viability by assessing plating efficiency on rich medium. WT, wild type. (E and F) Loss of Tor1 is epistatic over loss of Cds1. Strains were grown to log phase and shifted to 12 mM HU. The percentage of cells with septa was measured at the indicated times by staining with calcofluor and DAPI and visualized by fluorescence microscopy.
FIG. 5.
FIG. 5.
Tor1 positively regulates mitosis. (A) The Δtor1 mutation is synthetic lethal with cdc25-22. A diploid strain heterozygous for Δtor1 and cdc25-22 was subjected to meiosis and tetrad analysis. Plates were incubated at 25°C. (B) Overexpression of gad8+ rescues the synthetic lethality of tor1 cdc25-22. The same diploid strain as above was transformed with pIRT2-tor1+, pREP41-gad8+ (moderate overexpression), and pREP1-gad8+ (strong overexpression). Two double mutant spores containing each of the plasmids were isolated and streaked onto plates at 28°C (no viable spores were obtained with an empty vector). (C) The cdc2-Y15F mutation suppresses the elongated phenotype of cells lacking Tor1. Bars: 20 μm. (D) The Δtor1 mutation reverses the suppression of cdc25-22 by wee1-50. Cells from the indicated genotypes were streaked onto plates either at 28°C or 35°C (left panel), and cells were visualized by light microscopy (right panel). Bars: 20 μm. (E) The wee1-50 mutation partially suppresses the elongated phenotype of cells lacking Tor1. Cells were grown to mid-log phase at 30°C, photographed, and subjected to FACS analysis.
FIG. 6.
FIG. 6.
Tor1 is required for activation of Cdc2 after release from HU arrest. (A) Wild-type and Δtor1 cells were treated with 12 mM HU for 3.5 h, washed, and resuspended in fresh yeast extract. Samples from the indicated time points were taken for septation index measurement and Western blot analysis. (B) A working model. In response to DNA damage or DNA replication stress, Rad3 activates Chk1 or Cds1, respectively, leading to a delay in mitotic entry. In parallel, Rad3 keeps Tor1 inactive until DNA replication is completed. Regulation of Tor1 activity is not essential to prevent premature entry into mitosis but is required for reentry upon recovery from checkpoint arrest.
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