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The Ku subunit of telomerase binds Sir4 to recruit telomerase to lengthen telomeres in S. cerevisiae.

ABSTRACT: In Saccharomyces cerevisiae and in humans, the telomerase RNA subunit is bound by Ku, a ring-shaped protein heterodimer best known for its function in DNA repair. Ku binding to yeast telomerase RNA promotes telomere lengthening and telomerase recruitment to telomeres, but how this is achieved remains unknown. Using telomere-length analysis and chromatin immunoprecipitation, we show that Sir4 - a previously identified Ku-binding protein that is a component of telomeric silent chromatin - is required for Ku-mediated telomere lengthening and telomerase recruitment. We also find that specifically tethering Sir4 directly to Ku-binding-defective telomerase RNA restores otherwise-shortened telomeres to wild-type length. These findings suggest that Sir4 is the telomere-bound target of Ku-mediated telomerase recruitment and provide one mechanism for how the Sir4-competing Rif1 and Rif2 proteins negatively regulate telomere length in yeast.

SUBMITTER: Hass EP

PROVIDER: S-EPMC4547093 | biostudies-literature |

REPOSITORIES: biostudies-literature

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The Saccharomyces cerevisiae telomerase subunit Est3 binds telomeres in a cell cycle- and Est1-dependent manner and interacts directly with Est1 in vitro.

Project description:Telomerase is a telomere dedicated reverse transcriptase that replicates the very ends of eukaryotic chromosomes. Saccharomyces cerevisiae telomerase consists of TLC1 (the RNA template), Est2 (the catalytic subunit), and two accessory proteins, Est1 and Est3, that are essential in vivo for telomerase activity but are dispensable for catalysis in vitro. Est1 functions in both recruitment and activation of telomerase. The association of Est3 with telomeres occurred largely in late S/G2 phase, the time when telomerase acts and Est1 telomere binding occurs. Est3 telomere binding was Est1-dependent. This dependence is likely due to a direct interaction between the two proteins, as purified recombinant Est1 and Est3 interacted in vitro. Est3 abundance was neither cell cycle-regulated nor Est1-dependent. Est3 was the most abundant of the three Est proteins (84.3±13.3 molecules per cell versus 71.1±19.2 for Est1 and 37.2±6.5 for Est2), so its telomere association and/or activity is unlikely to be limited by its relative abundance. Est2 and Est1 telomere binding was unaffected by the absence of Est3. Taken together, these data indicate that Est3 acts downstream of both Est2 and Est1 and that the putative activation function of Est1 can be explained by its role in recruiting Est3 to telomeres.

| S-EPMC3088721 | biostudies-literature

The Shelterin TIN2 Subunit Mediates Recruitment of Telomerase to Telomeres.

Project description:Dyskeratosis Congenita (DC) is a heritable multi-system disorder caused by abnormally short telomeres. Clinically diagnosed by the mucocutaneous symptoms, DC patients are at high risk for bone marrow failure, pulmonary fibrosis, and multiple types of cancers. We have recapitulated the most common DC-causing mutation in the shelterin component TIN2 by introducing a TIN2-R282H mutation into cultured telomerase-positive human cells via a knock-in approach. The resulting heterozygous TIN2-R282H mutation does not perturb occupancy of other shelterin components on telomeres, result in activation of telomeric DNA damage signaling or exhibit other characteristics indicative of a telomere deprotection defect. Using a novel assay that monitors the frequency and extension rate of telomerase activity at individual telomeres, we show instead that telomerase elongates telomeres at a reduced frequency in TIN2-R282H heterozygous cells; this recruitment defect is further corroborated by examining the effect of this mutation on telomerase-telomere co-localization. These observations suggest a direct role for TIN2 in mediating telomere length through telomerase, separable from its role in telomere protection.

| S-EPMC4521702 | biostudies-literature

Telomerase and Tel1p preferentially associate with short telomeres in S. cerevisiae.

Project description:In diverse organisms, telomerase preferentially elongates short telomeres. We generated a single short telomere in otherwise wild-type (WT) S. cerevisiae cells. The binding of the positive regulators Ku and Cdc13p was similar at short and WT-length telomeres. The negative regulators Rif1p and Rif2p were present at the short telomere, although Rif2p levels were reduced. Two telomerase holoenzyme components, Est1p and Est2p, were preferentially enriched at short telomeres in late S/G2 phase, the time of telomerase action. Tel1p, the yeast ATM-like checkpoint kinase, was highly enriched at short telomeres from early S through G2 phase and even into the next cell cycle. Nonetheless, induction of a single short telomere did not elicit a cell-cycle arrest. Tel1p binding was dependent on Xrs2p and required for preferential binding of telomerase to short telomeres. These data suggest that Tel1p targets telomerase to the DNA ends most in need of extension.

| S-EPMC2650483 | biostudies-literature

Localization of telomeres and telomere-associated proteins in telomerase-negative Saccharomyces cerevisiae.

Project description:Cells lacking telomerase cannot maintain their telomeres and undergo a telomere erosion phase leading to senescence and crisis in which most cells become nonviable. On rare occasions survivors emerge from these cultures that maintain their telomeres in alternative ways. The movement of five marked telomeres in Saccharomyces cerevisiae was followed in wild-type cells and through erosion, senescence/crisis and eventual survival in telomerase-negative (est2::HYG) yeast cells. It was found that during erosion, movements of telomeres in est2::HYG cells were indistinguishable from wild-type telomere movements. At senescence/crisis, however, most cells were in G(2) arrest and the nucleus and telomeres traversed back and forth across the bud neck, presumably until cell death. Type I survivors, using subtelomeric Y' amplification for telomere maintenance, continued to show this aberrant telomere movement. However, Type II survivors, maintaining telomeres by a sudden elongation of the telomere repeats, became indistinguishable from wild-type cells, consistent with growth properties of the two types of survivors. When telomere-associated proteins Sir2p, Sir3p and Rap1p were tagged, the same general trend was seen-Type I survivors retained the senescence/crisis state of protein localization, while Type II survivors were restored to wild type.

| S-EPMC2784495 | biostudies-literature

Combining conservation and species-specific differences to determine how human telomerase binds telomeres.

Project description:Telomerase catalyzes telomeric DNA synthesis at chromosome ends to allow for continued cell division. The telomeric protein TPP1 is essential for enhancing the processivity of telomerase and recruiting the enzyme to telomeres. The telomerase interaction surface on human TPP1 has been mapped to 2 regions of the N-terminal oligosaccharide/oligonucleotide-binding (OB) domain, namely the TPP1 glutamate (E) and leucine (L)-rich (TEL) patch and the N terminus of TPP1-oligosaccharide/oligonucleotide-binding (NOB) region. To map the telomerase side of the interface, we exploited the predicted structural similarities for human and Tetrahymena thermophila telomerase as well as the species specificity of human and mouse telomerase for their cognate TPP1 partners. We show that swapping in the telomerase essential N-terminal (TEN) and insertions in fingers domain (IFD)-TRAP regions of the human telomerase catalytic protein subunit TERT into the mouse TERT backbone is sufficient to bias the species specificity toward human TPP1. Employing a structural homology-based mutagenesis screen focused on surface residues of the TEN and IFD regions, we identified TERT residues that are critical for contacting TPP1 but dispensable for other aspects of telomerase structure or function. We present a functionally validated structural model for how human telomerase engages TPP1 at telomeres, setting the stage for a high-resolution structure of this interface.

| S-EPMC6936698 | biostudies-literature

Telomerase subunit TERT regulates MYC stability independent of its role on telomeres

Project description:This SuperSeries is composed of the SubSeries listed below.

2015-04-23 | GSE60224 | GEO

Telomerase subunit TERT regulates MYC stability independent of its role on telomeres (array)

Project description:Constitutively active MYC and reactivated telomerase often co-exist in cancers. While the reactivation of telomerase is thought to be essential for replicative immortality, MYC, in conjunction with co-factors, confers several growth advantages to cancer cells. However, it is unclear which co-factors sustain elevated MYC activity in tumors . Here, we identify TERT, the catalytic subunit of telomerase, as a novel regulator of MYC stability in cancers. Binding of TERT to MYC stabilizes its levels on chromatin, contributing to either activation or repression of its target genes. Mechanistically, TERT regulates MYC ubiquitination and stability, and this effect of TERT is independent of its role on telomeres. Genetic inhibition and knocking out of TERT phenocopied the loss of MYC, resulting in reduced disease burden of early- and late-stage MYC-driven murine lymphomas. Conversly, the ectopic expression of TERT could substitute for reduced MYC in these functions. Finally we show that TERT null mice, unlike Terc null mice, show delayed onset of MYC induced lymphomagenesis. Accordingly, inhibiting TERT function in primary human leukemia cells blocked the expression of MYC targets, while Terc depletion had no effects . Based on our data, we conclude that the re-expression of TERT, a direct MYC target in tumors, provides a feed-forward mechanism to potentiate MYC-dependent oncogenesis. P493 cells were stably infected with the following viruses: pLKO shControl and pLKO shTERT.

2015-04-23 | E-GEOD-60175 | biostudies-arrayexpress

Telomerase subunit TERT regulates MYC stability independent of its role on telomeres (array)

2015-04-23 | GSE60175 | GEO

Repair of UV-induced DNA lesions in natural Saccharomyces cerevisiae telomeres is moderated by Sir2 and Sir3, and inhibited by yKu-Sir4 interaction.

Project description:Ultraviolet light (UV) causes DNA damage that is removed by nucleotide excision repair (NER). UV-induced DNA lesions must be recognized and repaired in nucleosomal DNA, higher order structures of chromatin and within different nuclear sub-compartments. Telomeric DNA is made of short tandem repeats located at the ends of chromosomes and their maintenance is critical to prevent genome instability. In Saccharomyces cerevisiae the chromatin structure of natural telomeres is distinctive and contingent to telomeric DNA sequences. Namely, nucleosomes and Sir proteins form the heterochromatin like structure of X-type telomeres, whereas a more open conformation is present at Y'-type telomeres. It is proposed that there are no nucleosomes on the most distal telomeric repeat DNA, which is bound by a complex of proteins and folded into higher order structure. How these structures affect NER is poorly understood. Our data indicate that the X-type, but not the Y'-type, sub-telomeric chromatin modulates NER, a consequence of Sir protein-dependent nucleosome stability. The telomere terminal complex also prevents NER, however, this effect is largely dependent on the yKu-Sir4 interaction, but Sir2 and Sir3 independent.

| S-EPMC5416773 | biostudies-literature

Telomerase subunit TERT regulates MYC stability independent of its role on telomeres (ChIP-Seq)

Project description:Constitutively active MYC and reactivated telomerase often co-exist in cancers. While the reactivation of telomerase is thought to be essential for replicative immortality, MYC, in conjunction with co-factors, confers several growth advantages to cancer cells. However, it is unclear which co-factors sustain elevated MYC activity in tumours . Here, we identify TERT, the catalytic subunit of telomerase, as a novel regulator of MYC stability in cancers. Binding of TERT to MYC stabilizes its levels on chromatin, contributing to either activation or repression of its target genes. Mechanistically, TERT regulates MYC ubiquitination and stability, and this effect of TERT is independent of its role on telomeres. Genetic inhibition and knocking out of TERT phenocopied the loss of MYC, resulting in reduced disease burden of early- and late-stage MYC-driven murine lymphomas. Conversly, the ectopic expression of TERT could substitute for reduced MYC in these functions. Finally we show that TERT null mice, unlike Terc null mice, show delayed onset of MYC induced lymphomagenesis. Accordingly, inhibiting TERT function in primary human leukemia cells blocked the expression of MYC targets, while Terc depletion had no effects . Based on our data, we conclude that the re-expression of TERT, a direct MYC target in tumors, provides a feed-forward mechanism to potentiate MYC-dependent oncogenesis. ChIP was performed using anti-MYC (sc-764) in primary lymphoma cells from Eµ-Myc;Tert -/- mice, Eµ-Myc;Tert +/+ mice, or in P493 cells treated with shTert, P493 shTerc and shControl.

2015-04-23 | E-GEOD-60223 | biostudies-arrayexpress

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