Telomerase Recruitment Research Articles

Ccq1-Tpz1TPP1 interaction facilitates telomerase and SHREC association with telomeres in fission yeast.

Evolutionarily conserved shelterin complex is essential for telomere maintenance in the fission yeast Schizosaccharomyces pombe. Elimination of the fission yeast shelterin subunit Ccq1 causes progressive loss of telomeres due to the inability to recruit telomerase, activates the DNA damage checkpoint, and loses heterochromatin at telomere/subtelomere regions due to reduced recruitment of the heterochromatin regulator complex Snf2/histone deacetylase-containing repressor complex (SHREC). The shelterin subunit Tpz1(TPP1) directly interacts with Ccq1 through conserved C-terminal residues in Tpz1(TPP1), and tpz1 mutants that fail to interact with Ccq1 show telomere shortening, checkpoint activation, and loss of heterochromatin. While we have previously concluded that Ccq1-Tpz1(TPP1) interaction contributes to Ccq1 accumulation and telomerase recruitment based on analysis of tpz1 mutants that fail to interact with Ccq1, another study reported that loss of Ccq1-Tpz1(TPP1) interaction does not affect accumulation of Ccq1 or telomerase. Furthermore, it remained unclear whether loss of Ccq1-Tpz1(TPP1) interaction affects SHREC accumulation at telomeres. To resolve these issues, we identified and characterized a series of ccq1 mutations that disrupt Ccq1-Tpz1(TPP1) interaction. Characterization of these ccq1 mutants established that Ccq1-Tpz1(TPP1) interaction contributes to optimal binding of the Ccq1-SHREC complex, and is critical for Rad3(ATR)/Tel1(ATM)-dependent Ccq1 Thr93 phosphorylation and telomerase recruitment.

Involvement of SRSF11 in cell cycle-specific recruitment of telomerase to telomeres at nuclear speckles.

Telomerase, a unique ribonucleoprotein complex that contains the telomerase reverse transcriptase (TERT), the telomerase RNA component (TERC) and the TERC-binding protein dyskerin, is required for continued cell proliferation in stem cells and cancer cells. Here we identify SRSF11 as a novel TERC-binding protein that localizes to nuclear speckles, subnuclear structures that are enriched in pre-messenger RNA splicing factors. SRSF11 associates with active telomerase enzyme through an interaction with TERC and directs it to nuclear speckles specifically during S phase of the cell cycle. On the other hand, a subset of telomeres is shown to be constitutively present at nuclear speckles irrespective of cell cycle phase, suggesting that nuclear speckles could be the nuclear sites for telomerase recruitment to telomeres. SRSF11 also associates with telomeres through an interaction with TRF2, which facilitates translocation of telomerase to telomeres. Depletion of SRSF11 prevents telomerase from associating with nuclear speckles and disrupts telomerase recruitment to telomeres, thereby abrogating telomere elongation by telomerase. These findings suggest that SRSF11 acts as a nuclear speckle-targeting factor that is essential for telomerase association with telomeres through the interactions with TERC and TRF2, and provides a potential target for modulating telomerase activity in cancer.

Abstract 3785: Cdk1 coordinates telomere replication by regulating the temporal recruitment of telomerase and the CST complex

Abstract Telomeres are nucleoprotein structures that cap the ends of linear chromosomes. They ensure genomic stability and complete replication of the genome. The enzyme telomerase mediates telomere elongation. Considering that telomerase is up-regulated in about 90% of human cancers and telomerase-mediated telomere elongation is significantly deregulated in cancers, insights from our study can be used to target the telomere replication machinery for therapeutic intervention in cancer. In our study, we use budding yeast to understand the mechanism of telomere maintenance. In budding yeast, Cdk1 coordinates the cell cycle-dependent elongation of telomere by telomerase. Cdk1 phosphorylates the telomere-specific ssDNA binding protein, Cdc13, which promotes the recruitment of telomerase to the telomere and thereby, telomere elongation. Cdc13 is also an integral part of the CST (Cdc13-Stn1-Ten1) complex, which provides the protective cap at the telomere and inhibits telomere elongation by telomerase. Therefore, the telomere length homeostasis is balanced between these two telomerase-extendable and CST-unextendable states. However, the cellular signaling that regulates the switch between these two states is largely unknown. Here we show that Cdk1 phosphorylates Stn1 sequentially to that of Cdc13. Cdk1-dependant phosphorylation of Stn1 is essential for telomere maintenance in vivo, CST stability and the inhibition of telomerase at the telomeres. By controlling the timing of Cdc13 and Stn1 phosphorylation during cell cycle progression, Cdk1 coordinates the recruitment of telomerase holoenzyme and CST complex to the telomeres respectively and hence the switch between the telomerase-extendable and CST-unextendable states of the telomeres in budding yeast. With increasing evidence of human CST complex being involved in telomere length maintenance, the results from my study will provide insights into telomere homeostasis in humans and its disruption in cancers. Citation Format: Veena Gopalakrishnan, Chang-Ching Liu, Lai Fong Poon, Tingdong Yan, Shang Li. Cdk1 coordinates telomere replication by regulating the temporal recruitment of telomerase and the CST complex. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3785. doi:10.1158/1538-7445.AM2015-3785

The Shelterin TIN2 Subunit Mediates Recruitment of Telomerase to Telomeres.

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.

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

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.

Minishelterins separate telomere length regulation and end protection in fission yeast

The conserved shelterin complex is critical for chromosome capping and maintaining telomere length homeostasis. In fission yeast, shelterin is comprised of five proteins. Taz1, Rap1, and Poz1 function as negative regulators of telomere elongation, whereas Pot1 and Tpz1 are critical for end capping and telomerase recruitment. How the five proteins work together to safeguard chromosome ends and promote telomere length homeostasis is a matter of great interest. Using a combination of deletions, fusions, and tethers, we define key elements of shelterin important for telomere length regulation. Surprisingly, deletion of the entire Rap1 and Poz1 proteins does not impair telomere length regulation as long as a static bridge is provided between Taz1 and Tpz1. Cells harboring minishelterin display wild-type telomere length and intact subtelomeric silencing. However, protection against end fusions in G1 is compromised in the absence of Rap1. Our data reveal a remarkable plasticity in shelterin architecture and separate functions in length regulation and end protection.

Elucidating Telomerase Function in Human Tumor and Stem Cell

A current focus of my lab is to elucidate the role of telomerase in stem cell maintenance and tumorigenesis. Human pluripotent stem cells have active telomerase and therefore long-term renewal capacity, but most human somatic cells lack telomerase function and therefore have a limited capacity for renewal. Patients with telomerase deficiencies that cause bone marrow failure, aplastic anemia and pulmonary fibrosis have accelerated telomere shortening, which gives rise to these tissue failures. Opposite to this effect is the telomerase reactivation that underlies the proliferative immortality of most human cancers. Despite these strong implications for human health, we lack understanding of the molecular mechanisms by which human cells regulate telomerase activity to ensure tissue homeostasis and how its dysfunction can lead to tumorigenesis. The natural regulation of telomerase activity in human tissue and the impact of telomere shortening on untransformed human cells can only be studied in a primary human stem cell system. Until recently, technical limitations, especially the inefficiency of genetic manipulation, have impeded the use of human stem cells as research tools. We have overcome this by establishing the use of site-specific nucleases to efficiently genetically engineer human pluripotent stem cells (hPSCs). This technology allows us, for the first time, to investigate two key regulatory events of human telomerase function in a genetically defined human stem cell system: its transcriptional regulation and the recruitment of telomerase to telomeres.

A second essential function of the Est1-binding arm of yeast telomerase RNA.

The enzymatic ribonucleoprotein telomerase maintains telomeres in many eukaryotes, including humans, and plays a central role in aging and cancer. Saccharomyces cerevisiae telomerase RNA, TLC1, is a flexible scaffold that tethers telomerase holoenzyme protein subunits to the complex. Here we test the hypothesis that a lengthy conserved region of the Est1-binding TLC1 arm contributes more than simply Est1-binding function. We separated Est1 binding from potential other functions by tethering TLC1 to Est1 via a heterologous RNA-protein binding module. We find that Est1-tethering rescues in vivo function of telomerase RNA alleles missing nucleotides specifically required for Est1 binding, but not those missing the entire conserved region. Notably, however, telomerase function is restored for this condition by expressing the arm of TLC1 in trans. Mutational analysis shows that the Second Essential Est1-arm Domain (SEED) maps to an internal loop of the arm, which SHAPE chemical mapping and 3D modeling suggest could be regulated by conformational change. Finally, we find that the SEED has an essential, Est1-independent role in telomerase function after telomerase recruitment to the telomere. The SEED may be required for establishing telomere extendibility or promoting telomerase RNP holoenzyme activity.

Contributions of the TEL-patch Amino Acid Cluster on TPP1 to Telomeric DNA Synthesis by Human Telomerase

Telomere maintenance is a highly coordinated process, and its misregulation is linked to cancer and telomere-shortening syndromes. Recent studies have shown that the TEL-patch—a cluster of amino acids on the surface of the shelterin component TPP1—is necessary for the recruitment of telomerase to the telomere in human cells. However, there has been only basic biochemical analysis of the role of TPP1 in the telomerase recruitment process. Here we develop an in vitro assay to quantitatively measure the contribution of the TEL-patch to telomerase recruitment—binding and extension of the first telomeric repeat. We also demonstrate that the TEL-patch contributes to the translocation step of the telomerase reaction. Finally, our quantitative observations indicate that the TEL-patch stabilizes the association between telomerase and telomeric DNA substrates, providing a molecular explanation for its contributions to telomerase recruitment and action.

Non-Shelterin Telomere Genes Deregulation in Plasma Cell Disorders

Telomeres are essential structures that protect the ends of linear chromosomes. When telomeres become dysfunctional, critically short ends are sensed by the DNA repair machinery triggering senescence or apoptosis as well as creating interchromosomal fusions. Telomere structure and functions depend on the telomerase enzyme (hTERT, hTERC and DKC1) for elongation, on the sheterin complex that regulates telomere length (TL) and protects them against degradation and fusion, and on the non-shelterin complex. The latter is constituted by a set of multifunctional factors, including RPA1 and the MRE11-RAD50-NBS1 (MRN) complex, that facilitate telomerase-based telomere elongation. Particularly, RPA1 might be a crucial link between telomere homeostasis and the replication of chromosome ends as it regulate the telomerase action during the cell cycle. The role of MRN complex in telomere maintenance parallels its function in DNA repair. In telomerase-positive cells, a reduction of the MRN complex results in G-overhang shortening, suggesting MRN complex involvement in the recruitment or action of telomerase. Previous data from our group have provided the first evidence of modifications in the mRNA expression of shelterin members in plasma cell disorders (Panero et al, Mol Med 2010; 16: 471-8; Blood Cells Mol Dis 2014; 52: 134-9). The aim of the present study was to examine the expression profile of the non-shelterin complex: RPA1, RAD50, MRE11, NBS1 and DKC1 in patients with multiple myeloma (MM) and monoclonal gammopathy of undetermined significance (MGUS). Results were correlated with hTERT (telomerase catalytic subunit) mRNA levels, TL, and clinico-pathological characteristics of patients. Bone marrow samples from 70 cases: 35 MM (21 females; mean age: 67.9 years; range: 30-87 years; 35.3% stage III) and 35 MGUS (21 females; mean age: 68.7 years; range: 39-88 years) were studied. All patients gave informed consent and the study was approved by the Ethics Committee of our Institution. Gene expression was quantified by Real-time Quantitative PCR, using Taq-Man methodology, and TL measurements were performed by Terminal Restriction Fragments. A significant increase in the mean mRNA levels of all non-shelterin genes in patients with MM respect to cases with MGUS was observed (Table 1). In order to evaluate whether the expression of these factors was associated to telomerase activity, hTERT transcripts were also quantified. An upregulation of telomerase expression, with significant differences between entities was found (p=0.03). In MM, higher DKC1 mRNA levels (0.10±0.02) in patients who over-expressed telomerase compared to patients with low hTERT expression (0.04±0.004) (p=0.008) was observed. No association between gene expression and TL or clinical parameters was found. To the best of our knowledge, our findings show for the first time a global modification in the expression of non-shelterin genes in MM and MGUS, suggesting that their upregulation in MM may occur as a result of the increasing need for DNA repair and telomerase recruitment, contributing to the progression of the disease. In addition, DKC1 was related to hTERTlevels in MM patients, supporting the role that DKC1 plays in telomerase complex stabilization and telomere maintenance.Table 1Expression profile of non-shelterin genes in patients with plasma cell disordersGenesMGUS (X±ES)MM (X±ES)p valueDKC10.036±0.0030.052±0.0050.02RAD500.060±0.0070.237±0.0640.0001MRE110.277±0.0400.545±0.1140.003NBS10.448±0.0660.894±0.1480.02RPA10.391±0.0440.591±0.0820.02 DisclosuresNo relevant conflicts of interest to declare.

PP2A and Aurora differentially modify Cdc13 to promote telomerase release from telomeres at G2/M phase.

In yeast, the initiation of telomere replication at the late S phase involves in combined actions of kinases on Cdc13, the telomere binding protein. Cdc13 recruits telomerase to telomeres through its interaction with Est1, a component of telomerase. However, how cells terminate the function of telomerase at G2/M is still elusive. Here we show that the protein phosphatase 2A (PP2A) subunit Pph22 and the yeast Aurora kinase homologue Ipl1 coordinately inhibit telomerase at G2/M by dephosphorylating and phosphorylating the telomerase recruitment domain of Cdc13, respectively. While Pph22 removes Tel1/Mec1-mediated Cdc13 phosphorylation to reduce Cdc13-Est1 interaction, Ipl1-dependent Cdc13 phosphorylation elicits dissociation of Est1-TLC1, the template RNA component of telomerase. Failure of these regulations prevents telomerase from departing telomeres, causing perturbed telomere lengthening and prolonged M phase. Together our results demonstrate that differential and additive actions of PP2A and Aurora on Cdc13 limit telomerase action by removing active telomerase from telomeres at G2/M phase.

Direct single-stranded DNA binding by Teb1 mediates the recruitment of Tetrahymena thermophila telomerase to telomeres.

The eukaryotic reverse transcriptase telomerase copies its internal RNA template to synthesize telomeric DNA repeats at chromosome ends in balance with sequence loss during cell proliferation. Previous work has established several factors involved in telomerase recruitment to telomeres in yeast and mammalian cells; however, it remains unclear what determines the association of telomerase with telomeres in other organisms. Here we investigate the cell cycle dependence of telomere binding by each of the seven Tetrahymena thermophila telomerase holoenzyme proteins TERT, p65, Teb1, p50, p75, p45, and p19. We observed coordinate cell cycle-regulated recruitment and release of all of the subunits, including the telomeric-repeat DNA-binding subunit Teb1. Using domain truncation and mutagenesis approaches, we investigated which subunits govern the interaction of telomerase holoenzyme with telomeres. Our results show that Teb1 is critical for telomere interaction of other holoenzyme subunits and demonstrate that high-affinity Teb1 DNA-binding activity is necessary and sufficient for cell cycle-regulated telomere association. Overall, these and additional findings indicate that in the ciliate Tetrahymena, telomerase recruitment to telomeres requires direct binding to single-stranded DNA, unlike the indirect DNA recognition through telomere-bound proteins essential in yeast and mammalian cells.

Tpz1-Ccq1 and Tpz1-Poz1 interactions within fission yeast shelterin modulate Ccq1 Thr93 phosphorylation and telomerase recruitment.

In both fission yeast and humans, the shelterin complex plays central roles in regulation of telomerase recruitment, protection of telomeres against DNA damage response factors, and formation of heterochromatin at telomeres. While shelterin is essential for limiting activation of the DNA damage checkpoint kinases ATR and ATM at telomeres, these kinases are required for stable maintenance of telomeres. In fission yeast, Rad3ATR and Tel1ATM kinases are redundantly required for telomerase recruitment, since Rad3ATR/Tel1ATM-dependent phosphorylation of the shelterin subunit Ccq1 at Thr93 promotes interaction between Ccq1 and the telomerase subunit Est1. However, it remained unclear how protein-protein interactions within the shelterin complex (consisting of Taz1, Rap1, Poz1, Tpz1, Pot1 and Ccq1) contribute to the regulation of Ccq1 Thr93 phosphorylation and telomerase recruitment. In this study, we identify domains and amino acid residues that are critical for mediating Tpz1-Ccq1 and Tpz1-Poz1 interaction within the fission yeast shelterin complex. Using separation of function Tpz1 mutants that maintain Tpz1-Pot1 interaction but specifically disrupt either Tpz1-Ccq1 or Tpz1-Poz1 interaction, we then establish that Tpz1-Ccq1 interaction promotes Ccq1 Thr93 phosphorylation, telomerase recruitment, checkpoint inhibition and telomeric heterochromatin formation. Furthermore, we demonstrate that Tpz1-Poz1 interaction promotes telomere association of Poz1, and loss of Poz1 from telomeres leads to increases in Ccq1 Thr93 phosphorylation and telomerase recruitment, and telomeric heterochromatin formation defect. In addition, our studies establish that Tpz1-Poz1 and Tpz1-Ccq1 interactions redundantly fulfill the essential telomere protection function of the shelterin complex, since simultaneous loss of both interactions caused immediate loss of cell viability for the majority of cells and generation of survivors with circular chromosomes. Based on these findings, we suggest that the negative regulatory function of Tpz1-Poz1 interaction works upstream of Rad3ATR kinase, while Tpz1-Ccq1 interaction works downstream of Rad3ATR kinase to facilitate Ccq1 Thr93 phosphorylation and telomerase recruitment.

POT1a and Components of CST Engage Telomerase and Regulate Its Activity in Arabidopsis

Protection of Telomeres 1 (POT1) is a conserved nucleic acid binding protein implicated in both telomere replication and chromosome end protection. We previously showed that Arabidopsis thaliana POT1a associates with the TER1 telomerase RNP, and is required for telomere length maintenance in vivo. Here we further dissect the function of POT1a and explore its interplay with the CST (CTC1/STN1/TEN1) telomere complex. Analysis of pot1a null mutants revealed that POT1a is not required for telomerase recruitment to telomeres, but is required for telomerase to maintain telomere tracts. We show that POT1a stimulates the synthesis of long telomere repeat arrays by telomerase, likely by enhancing repeat addition processivity. We demonstrate that POT1a binds STN1 and CTC1 in vitro, and further STN1 and CTC1, like POT1a, associate with enzymatically active telomerase in vivo. Unexpectedly, the in vitro interaction of STN1 with TEN1 and POT1a was mutually exclusive, indicating that POT1a and TEN1 may compete for the same binding site on STN1 in vivo. Finally, unlike CTC1 and STN1, TEN1 was not associated with active telomerase in vivo, consistent with our previous data showing that TEN1 negatively regulates telomerase enzyme activity. Altogether, our data support a two-state model in which POT1a promotes an extendable telomere state via contacts with the telomerase RNP as well as STN1 and CTC1, while TEN1 opposes these functions.

The Shelterin Component TPP1 Is a Binding Partner and Substrate for the Deubiquitinating Enzyme USP7

The telomeric shelterin component TPP1 has critical functions in telomeric protein complex assembly and telomerase recruitment and regulation. Here we identify USP7 as a novel interacting protein of the oligonucleotide/oligosaccharide-binding fold of TPP1, which was previously known to recruit telomerase to telomeres. We identify amino acids in TPP1 and USP7 that are critical for their interaction and multiple lysines within TPP1 that are oligo-ubiquitinated and deubiquitinated by USP7. Mutational analysis indicated that human TPP1 does not require ubiquitination for telomere association in contrast to previous observations reported for mouse Tpp1. Ubiquitination of human TPP1 also had no detectable effects on known protein interactions of TPP1 with TIN2, POT1, the CTC1-STN1-TEN1 complex, and telomerase. However, the close proximity of USP7 and telomerase binding sites on TPP1 suggest possible cross-talks. In addition, we found that TPP1 is degraded in a proteasome-dependent manner. Prevention of TPP1 ubiquitination prolonged TPP1 half-life ∼ 2-fold from 45 to 90 min, and remarkably, proteasome inhibition prompted complete stability of TPP1. This indicates that the proteasome destabilizes TPP1 through both direct and indirect pathways possibly involving TPP1-interacting proteins. Altogether, our work identifies novel regulatory circuits that contribute to TPP1 stability and function.

Identification of human TERT elements necessary for telomerase recruitment to telomeres.

Human chromosomes terminate in telomeres, repetitive DNA sequences bound by the shelterin complex. Shelterin protects chromosome ends, prevents recognition by the DNA damage machinery, and recruits telomerase. A patch of amino acids, termed the TEL-patch, on the OB-fold domain of the shelterin component TPP1 is essential to recruit telomerase to telomeres. In contrast, the site on telomerase that interacts with the TPP1 OB-fold is not well defined. In this study, we identify separation-of-function mutations in the TEN-domain of human telomerase reverse transcriptase (hTERT) that disrupt the interaction of telomerase with TPP1 in vivo and in vitro but have very little effect on the catalytic activity of telomerase. Suppression of a TEN-domain mutation with a compensatory charge-swap mutation in the TEL-patch indicates that their association is direct. Our findings define the interaction interface required for telomerase recruitment to telomeres, an important step towards developing modulators of this interaction as therapeutics for human disease.

Abstract 2250: Role of single strand binding protein 1 in TERT recruitment to telomeres and in maintaining telomere G-overhangs

Abstract Telomeres cap the ends of all eukaryotic chromosomes from degradation or end-to-end fusions. Mammalian telomeres are maintained by telomerase which is a ribonucleoprotein complex that includes a reverse transcriptase catalytic unit called TERT and a functional telomerase RNA (TR or TERC), which acts as a template to maintain telomere length. Telomerase maintains telomere length, by overcoming incomplete lagging strand synthesis known as the “end replication problem” during DNA replication. Mammalian stem and cancer cells utilize telomerase to extend telomeric G-overhangs to partially (stem cells) or completely (tumor cells) maintain telomere ends. Cells expressing telomerase also have higher levels of single strand binding 1 (SSB1) protein, which has a known critical role in the DNA damage responses and double strand break repair specifically by homologous recombination. We have established that SSB1 binds specifically to G-strand telomeric DNA in vitro, and associates in vivo with telomeres both in human and mouse cell lines. Interestingly, SSB1 interacts with the TERT protein, and determines the amount of TERT interaction with telomeres. Deletion/depletion of SSB1 reduces TERT interaction with telomeres and leads to G-overhang loss, but has no effect on in vitro telomerase activity. We will discuss, how SSB1 is required for telomerase recruitment to telomeres to facilitate G-strand DNA extension and to maintain telomeres. Citation Format: Tej K. Pandita, Raj Pandita. Role of single strand binding protein 1 in TERT recruitment to telomeres and in maintaining telomere G-overhangs. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2250. doi:10.1158/1538-7445.AM2014-2250

Molecular inhibition of telomerase recruitment using designer peptides: an in silico approach

Telomere holds special mechanism for solving end repair problems and maintaining genomic stability. Protection of telomeres 1 (POT1) which belongs to shelterin family is identified as a key protein that recruits telomerase by interacting with telomere repeat binding factors (TRB1-3). Since, deciphering the mechanism through which POT assembles telomerase is of great interest, computational approaches have been undertaken to understand the mechanism in a well- developed model system – Arabidopsis thaliana. As a first step, an untraditional approach was mediated to locate the active site residues on modeled AtPOT1b protein by interaction studies using molecular docking. To keep in trend with the recent developments, peptide construction and validation was promoted as the next step via molecular dynamics simulation studies. Finally, the validated peptides based on propensity score was evaluated for its efficacy as a potent inhibitor for POT and TRB1-3 interactions. The best peptide, namely, (1-2-d) out of 30 designed peptides, was proved to be vital inhibitor by weakening the interacting complexes.

Genetic and molecular identification of three human TPP1 functions in telomerase action: recruitment, activation, and homeostasis set point regulation.

Telomere length homeostasis is essential for the long-term survival of stem cells, and its set point determines the proliferative capacity of differentiated cell lineages by restricting the reservoir of telomeric repeats. Knockdown and overexpression studies in human tumor cells showed that the shelterin subunit TPP1 recruits telomerase to telomeres through a region termed the TEL patch. However, these studies do not resolve whether the TPP1 TEL patch is the only mechanism for telomerase recruitment and whether telomerase regulation studied in tumor cells is representative of nontransformed cells such as stem cells. Using genome engineering of human embryonic stem cells, which have physiological telomere length homeostasis, we establish that the TPP1 TEL patch is genetically essential for telomere elongation and thus long-term cell viability. Furthermore, genetic bypass, protein fusion, and intragenic complementation assays define two distinct additional mechanisms of TPP1 involvement in telomerase action at telomeres. We demonstrate that TPP1 provides an essential step of telomerase activation as well as feedback regulation of telomerase by telomere length, which is necessary to determine the appropriate telomere length set point in human embryonic stem cells. These studies reveal and resolve multiple TPP1 roles in telomere elongation and stem cell telomere length homeostasis.

Telomerase Activation after Recruitment in Fission Yeast

SummaryCurrent models depict that telomerase recruitment equates to activation. Telomeric DNA-binding proteins and the telomerase accessory proteins coordinate the recruitment of telomerase to the ends of chromosomes in a telomere length- and cell-cycle-dependent manner [1, 2, 3, 4]. Recent studies have demonstrated that the telomeric protein TPP1 and its binding protein TIN2 are key proteins for both telomerase recruitment and processivity in mammalian cells [5, 6, 7]. Although the precise molecular mechanism of telomerase recruitment has not yet been established, targeted point mutations within the oligonucleotide/oligosaccharide-binding (OB)-fold domain of TPP1 have been shown to impair telomerase association and processivity [8, 9, 10]. In fission yeast, telomerase is recruited through an interaction between the telomerase subunit Est1 and Ccq1, a component of the Pot1-Tpz1 telomere complex (POT1-TPP1 orthologs) [11, 12, 13, 14, 15]. Here, we demonstrate that association of telomerase with telomeres does not engage activity. We describe a mutation of Tpz1 that causes critical telomere shortening despite telomeric accumulation of the telomerase catalytic subunit, Trt1. Furthermore, Est1-directed telomerase association with Ccq1 is transient, and the Est1-Ccq1 interaction does not remain the bridge between telomeres and telomerase. Rather, direct interaction of Trt1 with Tpz1 is critical for telomere elongation. Moreover, Ccq1, which has been well characterized as a telomerase recruiter, is also required for the activation of telomere-associated telomerase. Our findings reveal a layer of telomerase regulation that controls activity after recruitment.