Negative Regulator Of Telomere Length Research Articles

Genetic Dissection of Dyskeratosis Congenita-Causing Mutations in TINF2 Using Human Pluripotent and Hematopoietic Stem Cell Models

The length of telomeres, which cap the ends of linear chromosomes and provide genomic stability, is tightly regulated in adult stem cells. The telomere reserve in the stem cell population sets the replicative potential of its differentiated progeny. For this reason, abnormally short telomeres in stem cells restrict the number of cell divisions that their differentiated progenies can undergo, eventually resulting in stem cell depletion and tissue failure syndromes. Telomere biology disorders (TBDs) display a broad range of clinical features, age of onset, and severity, which are all correlated with the extent of abnormal telomere shortening. One such early-onset TBD is dyskeratosis congenita (DC), which is a bone marrow predisposition syndrome characterized by a mucocutaneous triad (oral leukoplakia, nail dystrophy, and abnormal skin pigmentation) as well as other conditions driven by premature tissue aging. The leading cause of death in DC patients is bone marrow failure and hematopoietic stem cell transplantation is the only definite intervention to restore hematopoiesis.TINF2, which encodes the TIN2 protein, is mutated in 12% of patients and thereby the second most frequently altered gene in DC cases. TIN2 is a member of the shelterin protein complex bridging the double-strand binding shelterin proteins TRF1/TRF2, and the TPP1/POT1 heterodimer. Such interactions implicate a complex role of TIN2 in telomere length regulation: First, TIN2 stabilizes TRF1, which is a negative regulator of telomere length. Secondly, TPP1, which recruits telomerase, strictly requires TIN2 for telomere elongation and maintenance. TINF2-DC mutations are uniformly heterozygous and localize to a 30 amino acid coding stretch in exon 6 called the ‘DC cluster’. TINF2-DC mutations usually arise de novo and result in an earlier disease onset, shorter telomeres, and a more severe manifestation compared to other heterozygous DC-causing mutations in genes such as TR and TERT, which encode the components of the telomerase enzyme.How TINF2-DC mutations cause telomere shortening is unknown. Specifically, whether telomere shortening is caused by reduced telomerase action at telomeres or by degradation of telomeric DNA remains unresolved as studies using different model systems report contrasting results. The discrepancy could be attributed to differences in the model systems used in the studies, highlighting the need for a genetically trackable, primary preclinical human model system.Here, we report the development of two novel endogenous, isogenic model systems to study TINF2-DC mutations. First, we generated human embryonic stem cells (hESCs) engineered to express the TINF2-DC T284R mutation from the endogenous locus, which recapitulated the short telomere phenotype observed in DC patients. Using this model, we identified a gene editing strategy that elongates telomeres in the mutant stem cells and eventually restores replicative potential of the differentiated cells. Next, we used a xenotransplantation model of donor-derived human hematopoietic stem cells (hHSCs) to test the effects of target gene modifications on telomere length and proliferative capacity in vivo. We demonstrate that our models robustly complement each other and offer direct insights into the disease mechanism as well as avenues to potential therapeutic approaches. [Display omitted] DisclosuresBertuch: Elixirgen Therapeutics: Consultancy; ImmunityBio: Current equity holder in publicly-traded company; NIH/NCI,: Research Funding; DOD: Research Funding; Hyundai Hope on Wheels: Research Funding.

Telomeric zinc-finger associated protein (TZAP) in cancer biology: friend or foe?

The new identified protein telomeric zinc-finger associated protein (TZAP) is a negative regulator of telomere length. Since telomere length and telomere maintenance mechanisms are essential to cancer progression, TZAP is considered a new player in cancer biology. Here we aimed to analyze TZAP using the Cancer Genome Atlas data in a Pan-Cancer approach. We gathering data from TCGA Pan-Cancer studies utilizing cBioPortal, GEPIA and UALCAN. In total we analyzed 33 types of cancer (n=9664) and their respective controls (n=711). TZAP is transcribed in all cancers but less than 5% of all tumors show any somatic changes. TZAP was downregulated in kidney chromophobe carcinoma, and upregulated in esophageal cancer, head and neck squamous cell carcinomas, kidney renal clear cell carcinoma and in liver hepatocellular carcinoma. Globally, TZAP expression is related to favorable prognosis, associated to better overall and disease-free survival. Looking to specific tumors, TZAP expression has a dual behavior. Its downregulation is associated with poor prognosis in cervical squamous cell carcinoma, in kidney renal clear cell carcinoma, kidney papillary cell carcinoma, lung adenocarcinoma and pancreas adenocarcinoma. On the contrary, in adrenocortical carcinoma, colon and rectal cancer, brain lower grade glioma and prostate adenocarcinoma the upregulation of TZAP is related with poor prognosis. TZAP expression has a positive correlation with TRF1 and TRF2 in normal tissue but not in cancer. Our analyses indicate that TZAP has an important role in oncology and may be considered as a potential biomarker.

Telomeric double-strand DNA-binding proteins DTN-1 and DTN-2 ensure germline immortality in Caenorhabditis elegans.

Telomeres are nucleoprotein complexes at the ends of chromosomes and are indispensable for the protection and lengthening of terminal DNA. Despite the evolutionarily conserved roles of telomeres, the telomeric double-strand DNA (dsDNA)-binding proteins have evolved rapidly. Here, we identified double-strand telomeric DNA-binding proteins (DTN-1 and DTN-2) in Caenorhabditis elegans as non-canonical telomeric dsDNA-binding proteins. DTN-1 and DTN-2 are paralogous proteins that have three putative MYB-like DNA-binding domains and bind to telomeric dsDNA in a sequence-specific manner. DTN-1 and DTN-2 form complexes with the single-strand telomeric DNA-binding proteins POT-1 and POT-2 and constitutively localize to telomeres. The dtn-1 and dtn-2 genes function redundantly, and their simultaneous deletion results in progressive germline mortality, which accompanies telomere hyper-elongation and chromosomal bridges. Our study suggests that DTN-1 and DTN-2 are core shelterin components in C. elegans telomeres that act as negative regulators of telomere length and are essential for germline immortality.

Shorter telomere length, higher telomerase activity in association with tankyrase gene polymorphism contribute to high-altitude pulmonary edema.

High-altitude pulmonary edema (HAPE) is a noncardiogenic form of pulmonary edema, which is induced upon exposure to hypobaric hypoxia at high altitude (HA). Hypobaric hypoxia generates reactive oxygen species that may damage telomeres and disturb normal physiological processes. Telomere complex comprises of multiple proteins, of which, tankyrase (TNKS) is actively involved in DNA damage repairs. We hence investigated the association of TNKS and telomeres with HAPE to delineate their potential role at HA. The study was performed in three groups, High-altitude pulmonary edema patients (HAPE-p, n = 200), HAPE-resistant sojourners (HAPE-r, n = 200) and highland permanent healthy residents (HLs, n = 200). Variants of TNKS were genotyped using polymerase chain reaction-restriction fragment length polymorphism. Plasma TNKS level was estimated using enzyme-linked immunosorbent assay, expression of TNKS and relative telomere length were assessed by reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and telomerase activity was assessed by the telomere repeat amplification protocol assay. TNKS poly-ADP ribosylates the telomere-repeat factor (TRF), which is a negative regulator of telomere length. Consequently, TRF expression was also measured by RT-qPCR. The TNKS heterozygotes rs7015700GA were prevalent in HLs compared to the HAPE-p and HAPE-r. The plasma TNKS was significantly decreased in HAPE-p than HAPE-r (P =0.006). TNKS was upregulated 9.27 folds in HAPE-p (P = 1.01E-06) and downregulated in HLs by 3.3 folds (P = 0.02). The telomere length was shorter in HAPE-p compared to HAPE-r (P =0.03) and HLs (P= 4.25E-4). The telomerase activity was significantly higher in HAPE-p compared to both HAPE-r (P= 0.01) and HLs (P= 0.001). HAPE-p had the lowest TNKS levels (0.186 ± 0.031ng/μl) and the highest telomerase activity (0.0268 amoles/μl). The findings of the study indicate the association of TNKS and telomeres with HA adaptation/maladaptation.

Abstract 1468: ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator

Abstract Telomeres consist of repetitive TTAGGG repeats and together with the shelterin complex protect the ends of linear chromosomes from DNA repair mechanisms. Due to telomere shortening with every cell division proliferating cells need to ensure telomere maintenance by telomerase or in about 15% of cancer cells via the recombination based alternative lengthening of telomeres. Recently ZBTB48 was identified as a novel telomere-associated factor throughout the vertebrate lineage. Here we show that ZBTB48 binds directly to telomeres in vitro and in vivo, independently of the telomere maintenance mechanism. Loss of the protein leads to longer telomeres indicating a role as negative regulator of telomere length. Furthermore RNAseq, ChIPseq and quantitative expression proteomics demonstrate a function as transcriptional activator for a small set of target genes, including mitochondrial fission process 1 (MTFP1). This regulatory role links its telomeric function to mitochondrial metabolism and might play a role in cancer development. In summary ZBTB48 is a novel telomere binding protein which regulates telomere length and transcriptional activity. Citation Format: Arne Jahn, Grishma Rane, Maciej Paszkowski-Rogacz, Sergi Sayols, Alina Bluhm, Chung-Ting Han, Irena Draškovič, Arturo Londoño, Alan Prem Kumar, Frank Buchholz, Falk Butter, Dennis Kappei. ZBTB48 is both a vertebrate telomere-binding protein and a transcriptional activator [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1468.

Altered expression of telomere-associated genes in leukocytes among BRCA1 and BRCA2 carriers.

Telomere dysfunction resulting from telomere shortening and deregulation of shelterin components has been linked to the pathogenesis of age-related disorders, including cancer. Recent evidence suggests that BRCA1/2 (BRCA1 and BRCA2) tumor suppressor gene products play an important role in telomere maintenance. Although telomere shortening has been reported in BRCA1/2 carriers, the direct effects of BRCA1/2 haploinsufficiency on telomere maintenance and predisposition to cancer development are not completely understood. In this study, we assessed the telomere-associated and telomere-proximal gene expression profiles in peripheral blood leukocytes from patients with a BRCA1 or BRCA2 mutation, compared to samples from sporadic and familial breast cancer individuals. We found that 25 genes, including TINF2 gene (a negative regulator of telomere length), were significantly differentially expressed in BRCA1 carriers. Leukocyte telomere length analysis revealed that BRCA1/2 carriers had relatively shorter telomeres than healthy controls. Further, affected BRCA1/2 carriers were well differentiated from unaffected BRCA1/2 carriers by the expression of telomere-proximal genes. Our results link BRCA1/2 haploinsufficiency to changes in telomere length, telomere-associated as well as telomere-proximal gene expression. Thus, this work supports the effect of BRCA1/2 haploinsufficiency in the biology underlying telomere dysfunction in cancer development. Future studies evaluating these findings will require a large study population.

ZBTB48 is both a vertebrate telomere‐binding protein and a transcriptional activator

Telomeres constitute the ends of linear chromosomes and together with the shelterin complex form a structure essential for genome maintenance and stability. In addition to the constitutive binding of the shelterin complex, other direct, yet more transient interactions are mediated by the CST complex and HOT1/HMBOX1, while subtelomeric variant repeats are recognized by NR2C/F transcription factors. Recently, the Kruppel‐like zinc finger protein ZBTB48/HKR3/TZAP has been described as a novel telomere‐associated factor in the vertebrate lineage. Here, we show that ZBTB48 binds directly both to telomeric and to subtelomeric variant repeat sequences. ZBTB48 is found at telomeres of human cancer cells regardless of the mode of telomere maintenance and it acts as a negative regulator of telomere length. In addition to its telomeric function, we demonstrate through a combination of RNAseq, ChIPseq and expression proteomics experiments that ZBTB48 acts as a transcriptional activator on a small set of target genes, including mitochondrial fission process 1 (MTFP1). This discovery places ZBTB48 at the interface of telomere length regulation, transcriptional control and mitochondrial metabolism.

Heregulin, a new interactor of the telosome/shelterin complex in human telomeres.

Telomere length, shape and function depend on a complex of six core telomere-associated proteins referred to as the telosome or shelterin complex. We here demonstrate that the isoform β2 of the heregulin family of growth factors (HRGβ2) is a novel interactor of the telosome/shelterin complex in human telomeres. Analysis of protein-protein interactions using a high-throughput yeast two-hybrid (Y2H) screen identified RAP1, the only telomere protein that is conserved from yeasts to mammals, as a novel interacting partner of HRGβ2. Deletion analysis of RAP1 revealed that the linker domain, a region previously suggested to recruit negative regulators of telomere length, interacts specifically with HRGβ2. Co-immunoprecipitation and imaging experiments demonstrated that, in addition to RAP1, HRGβ2 could associate with the RAP1-associated telomeric repeat binding factor 2 (TRF2). Deletion analysis of HRGβ2 confirmed that a putative nuclear localization signal (NLS) was necessary for nuclear HRGβ2 to exert a negative regulation of telomere length whereas the N-terminus (extracellular) amino acids of HRGβ2 were sufficient to interact with RAP1/TRF2 and promote telomere shortening. Taken together, our studies identify nuclear HRGβ2 as one of the previously unknown regulators predicted to be recruited by the RAP1 linker domain to negatively regulate telomere length in human cells. Our current findings reveal that a new, but likely not the last, unexpected visitor has arrived to the "telosome/shelterin town".

Abstract 4577: Telomere oligonucleotides induce DNA damage responses in cancer cells: Mechanism of action and novel nanocomplex delivery system

Abstract Telomeres, the ribonucleoprotein complexes found at the ends of eukaryotic chromosomes, protect genomic DNA from end-to-end fusions and degradation by nucleases. During each round of cell division, the ends of telomeres are incompletely replicated, resulting in a net loss of telomeric DNA. When critically shortened, telomeres lose their ability to protect chromosomes from sustaining damage, thereby triggering cellular senescence or apoptosis. In numerous cancer types, administration of an oligonucleotide homologous to 3′-telomere overhang (T-oligo) induces potent DNA damage responses, presumably by mimicking or inducing telomere DNA damage, resulting in cell cycle arrest, senescence, or apoptosis. Remarkably, T-oligo induces minimal side-effects in normal cells, and thus, is actively being investigated as a novel anticancer therapeutic. However, as an oligonucleotide, T-oligo's intrinsic instability makes it susceptible to degradation by intracellular and serum nucleases, which severely limits its clinical use. To develop T-oligo as an innovative, effective therapeutic drug, it was complexed with a novel cationic alpha helical peptide, PVBLG-8 (PVBLG-8). PVBLG has been shown to be highly effective in delivering plasmid DNA and siRNA to a variety of cell lines, however, it has not been investigated using oligonucleotides. Here, our studies focus on its mechanism of action, stabilization and improved delivery. We show that PVBLG effectively stabilizes T-oligo by forming a nanocomplex (TOP complex), and enhances its anticancer properties both in vitro and in vivo. In addition, we identify new proteins involved in T-oligo's mechanism of action. The uptake of T-oligo by MM-AN melanoma cells was confirmed by immunofluorescence, and fluorescence-activated cell sorting analysis indicated that the TOP complex increased uptake by 15-fold. In MM-AN cells, T-oligo complexed with the PVBLG (0.025-.25mg/ml) inhibited growth in vitro in a dose dependent manner by 4.5-8.3-fold, compared to 3-fold by T-oligo alone. Similar results were seen in H358 lung cancer cells. Preformed MM-AN tumors in immunodeficient mice treated with the T-oligo-PVBLG nanocomplex resulted in a 3-fold reduction in tumor volume compared with T-oligo alone. This reduction in tumor volume was associated with decreased vascular endothelial growth factor expression and induction of thrombospondin-1 expression and apoptosis. In addition, T-oligo treatment demonstrated downregulation of telomere repeat factor 1, a negative regulator of telomere length, in MU melanoma cells, indicating a role for TRF1 in T-oligo-induced DNA damage responses. In summary, our investigation further defines T-oligo's mechanism of action, and demonstrates the potential of a novel nanoparticle, the TOP complex, as an effective anticancer therapeutic. Citation Format: Luke M. Wojdyla, Srijayaprakash B. Uppada, Neelu Puri. Telomere oligonucleotides induce DNA damage responses in cancer cells: Mechanism of action and novel nanocomplex delivery system. [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 4577. doi:10.1158/1538-7445.AM2014-4577

Molecular Recognition in Complexes of TRF Proteins with Telomeric DNA

Telomeres are specialized nucleoprotein assemblies that protect the ends of linear chromosomes. In humans and many other species, telomeres consist of tandem TTAGGG repeats bound by a protein complex known as shelterin that remodels telomeric DNA into a protective loop structure and regulates telomere homeostasis. Shelterin recognizes telomeric repeats through its two major components known as Telomere Repeat-Binding Factors, TRF1 and TRF2. These two homologous proteins are therefore essential for the formation and normal function of telomeres. Indeed, TRF1 and TRF2 are implicated in a plethora of different cellular functions and their depletion leads to telomere dysfunction with chromosomal fusions, followed by apoptotic cell death. More specifically, it was found that TRF1 acts as a negative regulator of telomere length, and TRF2 is involved in stabilizing the loop structure. Consequently, these proteins are of great interest, not only because of their key role in telomere maintenance and stability, but also as potential drug targets. In the current study, we investigated the molecular basis of telomeric sequence recognition by TRF1 and TRF2 and their DNA binding mechanism. We used molecular dynamics (MD) to calculate the free energy profiles for binding of TRFs to telomeric DNA. We found that the predicted binding free energies were in good agreement with experimental data. Further, different molecular determinants of binding, such as binding enthalpies and entropies, the hydrogen bonding pattern and changes in surface area, were analyzed to decompose and examine the overall binding free energies at the structural level. With this approach, we were able to draw conclusions regarding the consecutive stages of sequence-specific association, and propose a novel aspartate-dependent mechanism of sequence recognition. Finally, our work demonstrates the applicability of computational MD-based methods to studying protein-DNA interactions.

The splicing factor U2AF65 stabilizes TRF1 protein by inhibiting its ubiquitin-dependent proteolysis

The human telomeric protein TRF1 is a component of the six-subunit protein complex shelterin, which provides telomere protection by organizing the telomere into a high-order structure. TRF1 functions as a negative regulator of telomere length by controlling the access of telomerase to telomeres. Thus, the cellular abundance of TRF1 at telomeres should be maintained and tightly regulated to ensure proper telomere function. Here, we identify U2 small nuclear ribonucleoprotein (snRNP) auxiliary factor 65 (U2AF65), an essentialpre-mRNA splicingfactor, as a novel TRF1-interacting protein. U2AF65 interacts with TRF1 in vitro and in vivo and is capable of stabilizing TRF1 protein by inhibiting its ubiquitin-dependent proteolysis. We also found that U2AF65 interferes with the interaction between TRF1 and Fbx4, an E3 ubiquitin ligase for TRF1. Depletion of endogenous U2AF65 expression by short interfering RNA (siRNA) reduced the stability of endogenous TRF1 whereas overexpression of U2AF65 significantly extended the half-life of TRF1. These findings demonstrate that U2AF65 plays a critical role in regulating the level of TRF1 through physical interaction and ubiquitin-mediated proteolysis. Hence, U2AF65 represents a new route for modulating TRF1 function at telomeres.

Coordinate regulation between expression levels of telomere‐binding proteins and telomere length in breast carcinomas

Telomere dysregulation occurs in both the in situ and invasive stages of many carcinomas, including breast. Knockout experiments have identified several telomere-associated proteins required for proper telomere function and maintenance, including telomere repeat-binding factor 1 and 2 (TRF1 and TRF2), protection of telomeres (POT1), and TRF1-interacting nuclear factor 2 (TIN2). Using telomere content assays and quantitative reverse transcription-polymerase chain reaction (RT-PCR), we examined the relationship between telomere length and the mRNA levels of telomere-associated proteins in breast tumors. The levels of TRF2, TRF1, TIN2, and POT1 mRNA, but not telomerase reverse transcriptase (TERT) RNA, are inversely correlated with telomere content in breast tumors. Significant associations were identified between the mRNA levels of TRF1, TIN2, and POT1; however, there were no significant associations with the mRNA levels of TRF2 or TERT. These associations suggest that a complex transcriptional program coordinately regulates the expression of these mRNAs. We examined the promoter regions of the telomere-associated proteins to identify transcription factors consistent with the observed patterns of presumed coordinate expression. We demonstrated in human breast cancer cell lines that expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRF1, TIN2, and POT1 are upregulated by tumor necrosis factor-α (TNF-α), suggesting activation of the nuclear factor kappa B transcription factor. These findings link telomere content in breast tumors to the coordinate expression of several telomere-associated proteins previously shown to be negative regulators of telomere length in cell lines. The results further suggest a possible link between the expressions of the telomere-associated proteins and mediators of stress and inflammation.Telomere content assays and quantitative RT-PCR demonstrate that the levels of TRF2, TRF1, TIN2, and POT1 mRNA, but not telomerase reverse transcriptase (TERT) RNA, are inversely correlated with telomere content in breast tumors. Within human breast cancer cell lines, expressions of TRF1, TIN2, and POT1 are upregulated by dexamethasone, suggesting activation of the glucocorticoid receptor, whereas TERT, TRF2, TRF1, TIN2, and POT1 are upregulated by TNF-α, suggesting activation of the NFκB transcription factor. These findings link telomere content in breast tumors to the expression of several telomere-associated proteins previously shown to be negative regulators of telomere length in cell lines and suggest a link between the expressions of the telomere-associated proteins and mediators of stress and inflammation.

Abstract C19: DNA demethylation induces upregulation of telomere repeat-containing RNA (TERRA) and downregulation of telomerase activity in human leukemia cells

Abstract Background and aim: Telomere elongation mechanisms have been proposed to be regulated by the epigenetic status of the telomeric chromatin. Telomere repeat-containing RNAs (TERRAs), which are RNAs that originate from telomeric DNA transcription, can associate with the telomeric chromatin, where they are proposed to function as negative regulators of telomere length based on their ability to act as potent inhibitors of telomerase in vitro. To elucidate the regulatory mechanism of telomere length in a chromatin-dependent manner, we analyzed factors that influence telomere/telomerase regulation in human leukemia cells treated with 5-azacytidine, a currently available demethylating agent. Methods: Reagents and cells: Human leukemia cell lines (U937, K562, and HL-60) were treated with 0.001 to 10 μM 5-azacytidine (Sigma) for 24, 48, and 72 h; 5-azacytidine was added every 24 h. The antiproliferative effect of 5-azacytidine was measured using the cell counting kit-8 (Wako Chemicals). Telomere, telomerase and TERRA: Telomere length and telomerase activity were measured as reported previously (Ohyashiki JH, Br J Cancer 2005; 92: 1942–1947). The telomeric localization of TERRA was determined by RNA-FISH with strand-specific telomeric DNA probes. Experiments were performed in denaturing conditions with or without RNase, and the number of TERRA signal-positive cells was measured. Methylation: Bisulfite genomic DNA sequencing was performed using primers designed to recognize both methylated and unmethylated forms of the p16/INK4a and p15/INK4b promoter regions. For the genome-wide screening of DNA methylation patterns, the Human Methylation27 BeadChip (Illumina) was used. Results: Demethylating effect of 5-azacytidine: The half maximal inhibitory concentration (IC50) of 5-azacytidine was 3.4 μM in U937, 3.6 μM in K562, and 1 μM in HL-60. Bisulfite genomic DNA sequencing revealed that the p15/INK4b promoter region was markedly demethylated in U937 cells after 72 h treatment with the IC50 dose of 5-azacytidine. Similarly, we noted genome-wide hypomethylation in 5-azacytidine-treated U937 cells using the Human Methylation27 BeadChip, whereas we noted only modest hypomethylation in 5-azacytidine-treated K562 and HL-60 cells. Upregulation of TERRA and telomerase inhibition: Based on the results obtained from the methylation analysis, we identified the localization of TERRA in 5-azacytidine-treated U937 and K562 cells. We observed 0∼2 dots corresponding to TERRA per cell in untreated U937 or K562 cells, and the signals were not detected in RNaseA-treated samples. In U937 cells, TERRA signals significantly increased in accordance with the decrease in telomerase activity. In contrast, neither an increase in TERRA signals nor a decrease in telomerase activity was observed in K562 cells. This indicates that the upregulation of TERRA and telomerase inhibition were closely related to genome-wide hypomethylation. Conclusion: There is emerging evidence for the clinical utility of demethylating agents, such as 5-azacytidine in myelodysplastic syndrome; however, the molecular mechanism of 5-azacytidine has not been fully elucidated. Our results indicate that 5-azacytidine induces the upregulation of TERRA, thereby inhibiting telomerase in U937 cells. Although there is still a long way to go before realizing the diagnostic and therapeutic uses of TERRA in leukemia patients, our study provided novel information on TERRA in the telomere and the telomerase biology of human leukemia. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C19.

TIN2 Protein Dyskeratosis Congenita Missense Mutants Are Defective in Association with Telomerase

Dyskeratosis congenita (DC) is a progressive and heterogeneous congenital disorder that affects multiple systems and is characterized by bone marrow failure and a triad of abnormal skin pigmentation, nail dystrophy, and oral leukoplakia. One common feature for all DC patients is abnormally short telomeres and defects in telomere biology. Most of the known DC mutations have been found to affect core components of the telomerase holoenzyme. Recently, multiple mutations in the gene encoding the telomeric protein TIN2 have been identified in DC patients with intact telomerase genes, but the molecular mechanisms underlying TIN2 mutation-mediated DC remain unknown. Here, we demonstrate that ectopic expression of TIN2 with DC missense mutations in human cells led to accelerated telomere shortening, similar to the telomere phenotypes found in DC patients. However, this telomere shortening was not accompanied by changes in total telomerase activity, localization of TIN2, or telomere end protection status. Interestingly, we found TIN2 to participate in the TPP1-dependent recruitment of telomerase activity. Furthermore, DC mutations in TIN2 led to its decreased ability to associate with TERC and telomerase activity. Taken together, our data suggest that TIN2 mutations in DC may compromise the telomere recruitment of telomerase, leading to telomere shortening and the associated pathogenesis.

Single- and double-stranded DNA binding proteins act in concert to conserve a telomeric DNA core sequence

BackgroundTelomeres are protective cap structures at the ends of the linear eukaryotic chromosomes, which provide stability to the genome by shielding from degradation and chromosome fusions. The cap consists of telomere-specific proteins binding to the respective single- and double-stranded parts of the telomeric sequence. In addition to the nucleation of the chromatin structure the telomere-binding proteins are involved in the regulation of the telomere length. However, the telomeric sequences are highly diverged among yeast species. During the evolution this high rate of divergency presents a challenge for the sequence recognition of the telomere-binding proteins.ResultsWe found that the Saccharomyces castellii protein Rap1, a negative regulator of telomere length, binds a 12-mer minimal binding site (MBS) within the double-stranded telomeric DNA. The sequence specificity is dependent on the interaction with two 5 nucleotide motifs, having a 6 nucleotide centre-to-centre spacing. The isolated DNA-binding domain binds the same MBS and retains the same motif binding characteristics as the full-length Rap1 protein. However, it shows some deviations in the degree of sequence-specific dependence in some nucleotide positions. Intriguingly, the positions of most importance for the sequence-specific binding of the full-length Rap1 protein coincide with 3 of the 4 nucleotides utilized by the 3' overhang binding protein Cdc13. These nucleotides are very well conserved within the otherwise highly divergent telomeric sequences of yeasts.ConclusionsRap1 and Cdc13 are two very distinct types of DNA-binding proteins with highly separate functions. They interact with the double-stranded vs. the single-stranded telomeric DNA via significantly different types of DNA-binding domain structures. However, we show that they are dependent on coinciding nucleotide positions for their sequence-specific binding to telomeric sequences. Thus, we conclude that during the molecular evolution they act together to preserve a core sequence of the telomeric DNA.

Molecular Properties of Telomeric TRF1/TRF2 - DNA Systems

Telomeres are nucleoprotein complexes that protect the ends of linear eukaryotic chromosomes from degradation and fusion. Human telomeric DNA contains tandem arrays of double stranded TTAGGG repeats. Telomeric DNA forms specific complexes with many different proteins (shelterins), among which TRF1 and TRF2 are the most essential for the maintenance of telomere structure and function. TRF1 is a negative regulator of telomere length whereas TRF2 is involved in formation of telomeric higher order structures (t-loops), and functions more related to capping the DNA end. Both proteins bind to DNA as pre-formed homodimers. Although cellular functions of both these proteins are different, their structures are very similar. Both TRF1 and TRF2 contain two conserved sequence motives which form specific domains, namely homodimerisation and Myb-DNA binding domains.In order to reveal the molecular properties of both proteins and also differences between binding modes of TRF1 and TRF2 to telomeric DNA, detailed studies of both binding domains have been performed. We carried out molecular dynamic simulations of TRF1 and TRF2 binding domains and their complexes with DNA. Starting models of studied systems were based on X-ray structures of TRF1 and TRF2 Myb-DNA binding domains [1]. The results have revealed structural differences between bound proteins and structural differences of their binding patterns with DNA. Additionally, we provide experimental evidence that interaction of both shelterins and DNA can be specifically perturbed by small molecular weight ligands. These results support the idea that TRF1/TRF2 - DNA systems are potential new targets for anticancer therapy.1. R. Court, L. Chapman, L. Fairall, D. Rhodes, EMBO Reports 6 (2005) 39-45.

TRF1 Controls Telomere Length and Mitotic Fidelity in Epithelial Homeostasis

TRF1 is a component of the shelterin complex at mammalian telomeres; however, a role for TRF1 in telomere biology in the context of the organism is unclear. In this study, we generated mice with transgenic TRF1 expression targeted to epithelial tissues (K5TRF1 mice). K5TRF1 mice have shorter telomeres in the epidermis than wild-type controls do, and these are rescued in the absence of the XPF nuclease, indicating that TRF1 acts as a negative regulator of telomere length by controlling XPF activity at telomeres, similar to what was previously described for TRF2-overexpressing mice (K5TRF2 mice). K5TRF1 cells also show increased end-to-end chromosomal fusions, multitelomeric signals, and increased telomere recombination, indicating an impact of TRF1 on telomere integrity, again similar to the case in K5TRF2 cells. Intriguingly, K5TRF1 cells, but not K5TRF2 cells, show increased mitotic spindle aberrations. TRF1 colocalizes with the spindle assembly checkpoint proteins BubR1 and Mad2 at mouse telomeres, indicating a link between telomeres and the mitotic spindle. Together, these results demonstrate that TRF1, like TRF2, negatively regulates telomere length in vivo by controlling the action of the XPF nuclease at telomeres; in addition, TRF1 has a unique role in the mitotic spindle checkpoint.

Analysis of Telomeric Proteins Expression in Plasma Cell Disorders

Telomeres are specialized DNA-protein complexes that play an essential role in maintaining chromosomal integrity and stability. Their progressive shortening with each cell division can be compensated by the enzyme telomerase, a specialized ribonucleoprotein complex that includes an RNA template (TERC) and a reverse transcriptase catalytic subunit (hTERT). In addition to telomerase, telomere length (TL) maintenance depends on the shelterin complex, a core of telomere-binding proteins that protect telomeric ends. Among them, TRF1 and TRF2 (telomeric repeat-binding factor 1 and 2) are negative regulators of TL. On the other hand, TANK1 (tankyrase-like protein 1) (positive regulator) interacts with and ADP-ribosylates TRF1, displacing it from telomeric DNA. Multiple myeloma (MM) is characterized by neoplastic proliferation of monoclonal plasma cells. The natural course of disease may progress through monoclonal gammopathy of undetermined significance (MGUS) to MM. In this study, we evaluated the association between the expression of telomeric genes TRF1, TRF2, TANK1 and hTERT in patients with MM and MGUS. Results were correlated with clinicopathologic characteristics of patients. Fifty-seven patients: 32 MM (21 males; mean age: 70.5 years; range: 52–86 years; 53% stage III) and 25 MGUS (9 males; mean age: 66.4 years; range: 39–82) were studied. All patients provided informed consent and the study was approved by the Ethics Committee of our Institution. RNA was extracted from bone marrow cells of patients and peripheral blood lymphocytes of controls. Gene expression was quantified by real time PCR, using Taq- Man methodology. GAPDH (Gliceraldehide-3-phosphate dehydrogenase) was used as housekeeping gene to normalize the expression of each target gene. The expression levels of TRF1, TRF2, TANK1 and hTERT mRNA were significantly different in cases respect to controls. In patients, lower levels of TRF1, with statistical differences between MM and MGUS, and higher expression of TRF2, TANK1 and hTERT were observed (Table 1). In both pathologies, significant correlation between TRF2 and hTERT were found (r=0.70; p=0.0001 and r=0.36; p=0.07, for MM and MGUS, respectively). Furthermore, a high significant association between TANK1 expression and that of TRF2 and hTERT in MM was observed (r=0.80, p=0.0001; r= 0.45, p=0.009, respectively).Table 1. Summary of the TRF1, TRF2, TANK1 and hTERT mRNA levels in MM and MGUSGroupTRF1 Mean±SETRF2 Mean±SETANK1 Mean±SEhTERT Mean±SENormal (n=9)14.3±10.90.0001±10−80.03±10−40MGUS (n=25)0.56±0.03*0.06±0.0001**0.28±0.001*1.60±0.3MM (n=32)0.18±0.003*,≠0.06±0.0001**0.27±0.0008**1.74±0.5Significant differences respect to controls: *p=0.0001; **p<0.01; Significant differences respect to MGUS: ≠p=0.03For a better analysis of hTERT expression, patients were divided into two groups: hTERT mRNA<1 (A) and >1 (B). In MM, the mean level of expression was: 0.04±0.001 for group A (n=21) and 7.5±4.9 for group B (n=11), whereas in MGUS, the mean values observed were 0.28±0.001 (n=17) and 5.8±0.78 (n=8), respectively (p=0.0001). Analysis of hTERT and clinical parameters showed a significant correlation in LDH (r=0.77; p=0.005) and creatinine (r=0.73; p=0.01) for MM group B, and in b2 microglobulin (r=0.74; p=0.05) for MGUS group B. Our findings showed an association between telomeric proteins and plasma cell disorders. Differences in TRF1 expression between MM and MGUS would suggest that this gene may play a role in evolution of MGUS to MM. Finally, the correlation between hTERT and clinical characteristics would indicate that the expression of this gene could be a possible prognostic factor in these pathologies.

Regulation of Telomeric Repeat Binding Factor 1 Binding to Telomeres by Casein Kinase 2-mediated Phosphorylation

Telomere maintenance is essential for continued cell proliferation and chromosome stability. Telomeres are maintained by telomerase and a collection of associated proteins. The telomeric protein telomeric repeat binding factor 1 (TRF1) negatively regulates telomere length by inhibiting access of telomerase at telomere termini. Here we report that TRF1 interacts with the beta subunit of casein kinase 2 (CK2) and serves as a substrate for CK2. CK2-mediated phosphorylation is required for the efficient telomere binding of TRF1 in vitro and in vivo. Inhibition of CK2 by the CK2 inhibitor 5,6-dichloro-1-beta-d-ribofuranosylbenzimidazole decreased the ability of TRF1 to bind telomeric DNA. The resulting telomere-unbound form of TRF1 was then ubiquitinated and degraded by the proteasome. Partial knockdown of CK2 by small interfering RNA resulted in removal of TRF1 from telomeres and subsequent degradation of TRF1. Mapping of the CK2 target site identified threonine 122 as a substrate in TRF1. A threonine to alanine change at this position led to a diminished DNA binding due to reduced dimerization of TRF1. In addition, phosphorylation of threonine 122 seemed critical for TRF1-mediated telomere length control. Our findings suggest that CK2-mediated phosphorylation of TRF1 plays an important role in modulating telomere length homeostasis by determining the levels of TRF1 at telomeres.

Sensitivity of Yeast Strains with Long G-Tails to Levels of Telomere-Bound Telomerase

The Saccharomyces cerevisiae Pif1p helicase is a negative regulator of telomere length that acts by removing telomerase from chromosome ends. The catalytic subunit of yeast telomerase, Est2p, is telomere associated throughout most of the cell cycle, with peaks of association in both G1 phase (when telomerase is not active) and late S/G2 phase (when telomerase is active). The G1 association of Est2p requires a specific interaction between Ku and telomerase RNA. In mutants lacking this interaction, telomeres were longer in the absence of Pif1p than in the presence of wild-type PIF1, indicating that endogenous Pif1p inhibits the active S/G2 form of telomerase. Pif1p abundance was cell cycle regulated, low in G1 and early S phase and peaking late in the cell cycle. Low Pif1p abundance in G1 phase was anaphase-promoting complex dependent. Thus, endogenous Pif1p is unlikely to act on G1 bound Est2p. Overexpression of Pif1p from a non-cell cycle-regulated promoter dramatically reduced viability in five strains with impaired end protection (cdc13–1, yku80Δ, yku70Δ, yku80–1, and yku80–4), all of which have longer single-strand G-tails than wild-type cells. This reduced viability was suppressed by deleting the EXO1 gene, which encodes a nuclease that acts at compromised telomeres, suggesting that the removal of telomerase by Pif1p exposed telomeres to further C-strand degradation. Consistent with this interpretation, depletion of Pif1p, which increases the amount of telomere-bound telomerase, suppressed the temperature sensitivity of yku70Δ and cdc13–1 cells. Furthermore, eliminating the pathway that recruits Est2p to telomeres in G1 phase in a cdc13–1 strain also reduced viability. These data suggest that wild-type levels of telomere-bound telomerase are critical for the viability of strains whose telomeres are already susceptible to degradation.