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ABSTRACT: Human telomere function is mediated by shelterin, a six-subunit complex that is required for telomere replication, protection, and cohesion. TIN2, the central component of shelterin, has binding sites to three subunits: TRF1, TRF2, and TPP1. Here we identify a fourth partner, heterochromatin protein 1γ (HP1γ), that binds to a conserved canonical HP1-binding motif, PXVXL, in the C-terminal domain of TIN2. We show that HP1γ localizes to telomeres in S phase, where it is required to establish/maintain cohesion. We further demonstrate that the HP1-binding site in TIN2 is required for sister telomere cohesion and can impact telomere length maintenance by telomerase. Remarkably, the PTVML HP1-binding site is embedded in the recently identified cluster of mutations in TIN2 that gives rise to dyskeratosis congenita (DC), an inherited bone marrow failure syndrome caused by defects in telomere maintenance. We show that DC-associated mutations in TIN2 abrogate binding to HP1γ and that DC patient cells are defective in sister telomere cohesion. Our data indicate a novel requirement for HP1γ in the establishment/maintenance of cohesion at human telomeres and, furthermore, may provide insight into the mechanism of pathogenesis in TIN2-mediated DC.
Genes & development 08/2011; 25(17):1807-19. · 12.08 Impact Factor
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ABSTRACT: The Ku heterodimer associates with the Saccharomyces cerevisiae telomere, where it impacts several aspects of telomere structure and function. Although Ku avidly binds DNA ends via a preformed channel, its ability to associate with telomeres via this mechanism could be challenged by factors known to bind directly to the chromosome terminus. This has led to uncertainty as to whether Ku itself binds directly to telomeric ends and whether end association is crucial for Ku's telomeric functions. To address these questions, we constructed DNA end binding-defective Ku heterodimers by altering amino acid residues in Ku70 and Ku80 that were predicted to contact DNA. These mutants continued to associate with their known telomere-related partners, such as Sir4, a factor required for telomeric silencing, and TLC1, the RNA component of telomerase. Despite these interactions, we found that the Ku mutants had markedly reduced association with telomeric chromatin and null-like deficiencies for telomere end protection, length regulation, and silencing functions. In contrast to Ku null strains, the DNA end binding defective Ku mutants resulted in increased, rather than markedly decreased, imprecise end-joining proficiency at an induced double-strand break. This result further supports that it was the specific loss of Ku's telomere end binding that resulted in telomeric defects rather than global loss of Ku's functions. The extensive telomere defects observed in these mutants lead us to propose that Ku is an integral component of the terminal telomeric cap, where it promotes a specific architecture that is central to telomere function and maintenance.
PLoS Genetics 08/2011; 7(8):e1002233. · 8.69 Impact Factor
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ABSTRACT: Since 1998, there have been great advances in our understanding of the pathogenesis of dyskeratosis congenita (DC), a rare inherited bone marrow failure and cancer predisposition syndrome with prominent mucocutaneous abnormalities and features of premature aging. DC is now characterized molecularly by the presence of short age-adjusted telomeres. Mutations in seven genes have been unequivocally associated with DC, each with a role in telomere length maintenance. These observations, combined with knowledge that progressive telomere shortening can impose a proliferative barrier on dividing cells and contribute to chromosome instability, have led to the understanding that extreme telomere shortening drives the clinical features of DC. However, some of the genes implicated in DC encode proteins that are also components of H/ACA-ribonucleoprotein enzymes, which are responsible for the post-translational modification of ribosomal and spliceosomal RNAs, raising the question whether alterations in these activities play a role in the pathogenesis of DC. In addition, recent reports suggest that some cases of DC may not be characterized by short age-adjusted telomeres. This review will highlight our current knowledge of the telomere length defects in DC and the factors involved in its development.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis 07/2011; 730(1-2):43-51. · 2.85 Impact Factor
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ABSTRACT: Telomere biology disorders are a complex set of illnesses defined by the presence of very short telomeres. Individuals with classic dyskeratosis congenita have the most severe phenotype, characterized by the triad of nail dystrophy, abnormal skin pigmentation, and oral leukoplakia. More significantly, these individuals are at very high risk of bone marrow failure, cancer, and pulmonary fibrosis. A mutation in one of six different telomere biology genes can be identified in 50–60% of these individuals. DKC1, TERC, TERT, NOP10, and NHP2 encode components of telomerase or a telomerase-associated factor and TINF2, a telomeric protein. Progressively shorter telomeres are inherited from generation to generation in autosomal dominant dyskeratosis congenita, resulting in disease anticipation. Up to 10% of individuals with apparently acquired aplastic anemia or idiopathic pulmonary fibrosis also have short telomeres and mutations in TERC or TERT. Similar findings have been seen in individuals with liver fibrosis or acute myelogenous leukemia. This report reviews basic aspects of telomere biology and telomere length measurement, and the clinical and genetic features of those disorders that constitute our current understanding of the spectrum of illness caused by defects in telomere biology. We also suggest a grouping schema for the telomere disorders.
Genetics in medicine: official journal of the American College of Medical Genetics 12/2010; 12(12):753-64. · 3.92 Impact Factor
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Aging 11/2010; 2(11):756-7. · 5.13 Impact Factor
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ABSTRACT: The fourth AACR Special Conference on The Role of Telomeres and Telomerase in Cancer Research was held February 27 to March 2, 2010 in Fort Worth, TX. The meeting was organized to bring together those interested in the basic molecular mechanisms that govern telomere dynamics and stability with those interested in the clinical implications of telomere dysfunction and the use of telomeres and telomerase as therapeutic targets. The meeting was extremely successful as evidenced by the attendance and quality of the presentations. Indeed, several important themes emerged including (a) the intricate connection between the DNA replication and repair machineries in basic telomere replication and stability, (b) the complex interplay between the telomere-specific shelterin components and DNA repair proteins, (c) the nontelomeric functions of TERT in numerous cell types including stem cells, (d) a growing appreciation for the connection that exists between telomere maintenance deficiency states and diverse conditions such as idiopathic pulmonary fibrosis and hematopoietic malignancies, and (e) the successful progression of agents targeting telomerase directly and immunologically to phase III clinical trials. Evident at the meeting was the vibrant energy that permeates the telomere field and the important biological and medical findings that it continues to yield.
Cancer Research 10/2010; 70(19):7365-71. · 7.86 Impact Factor
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PhD Jason T. Yustein MD,
Surya Rednam MD,
PhD Alison A. Bertuch MD,
John A. Goss MD,
Mary L. Brandt MD,
Karen Eldin MD,
Xinyan Lu PhD,
John Hicks MD, PhD, DDS,
Jason T. Yustein,
Surya Rednam, Alison A. Bertuch,
John A. Goss,
Mary L. Brandt,
Karen Eldin,
Xinyan Lu,
John Hicks
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ABSTRACT: We describe a male with a large abdominal mass, most likely originating from the liver, with capsule rupture and tumor dissemination into the abdominal cavity. Adherence of the tumor to the diaphragm and lower right colon also were noted. A comprehensive evaluation of the mass revealed no tumor-defining histopathologic, immunocytochemical, ultrastructural, cytogenetic, or translocation features. The malignant tumor was found to have a novel translocation (X;19)(q13;13.3), which has not been reported in small round cell tumors of childhood or adults. The final diagnosis rendered was an undifferentiated small round cell tumor of uncertain cell of origin. Pediatr Blood Cancer 2010;54:1041–1044 © 2010 Wiley-Liss, Inc.
Pediatric Blood & Cancer 06/2010; 54(7):1041 - 1044. · 1.89 Impact Factor
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Terzah M Horton,
Gaye Jenkins,
Debananda Pati,
Linna Zhang,
M Eileen Dolan,
Albert Ribes-Zamora, Alison A Bertuch,
Susan M Blaney,
Shannon L Delaney,
Madhuri Hegde,
Stacey L Berg
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ABSTRACT: The poly(ADP-ribose) polymerase (PARP) inhibitor ABT-888 potentiates the antitumor activity of temozolomide (TMZ). TMZ resistance results from increased O(6)-methylguanine-DNA methyltransferase (MGMT) activity and from mismatch repair (MMR) system mutations. We evaluated the relative importance of MGMT activity, MMR deficiency, nonhomologous end joining (NHEJ), and PARP activity in ABT-888 potentiation of TMZ. MMR-proficient and MMR-deficient leukemia cells with varying MGMT activity, as well as primary leukemia samples, were used to determine TMZ IC(50) alone and with ABT-888. ABT-888 effectively inhibited PARP activity and enhanced TMZ growth inhibition in most leukemia cells. ABT-888 potentiation was most effective in MMR-deficient cells with low MGMT activity [potentiation factor (PF) = 21]. ABT-888 also potentiated TMZ activity in MMR-deficient cells with elevated MGMT activity. Unexpectedly, ABT-888 also enhanced TMZ activity in MMR-proficient cells (PF = 3-7). ABT-888 potentiation was unrelated to NHEJ activity. ABT-888 potentiated TMZ (PF = 2-5) in two of four acute myeloid leukemia patient samples but showed little potentiation in primary acute lymphoblastic leukemia. In conclusion, although ABT-888 potentiation of TMZ was most pronounced in MMR-deficient cells with low MGMT activity, neither MMR proficiency nor MGMT overexpression completely abrogated ABT-888 potentiation of TMZ.
Molecular Cancer Therapeutics 09/2009; 8(8):2232-42. · 5.23 Impact Factor
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ABSTRACT: In yeast, as in humans, telomere length varies among individuals and is controlled by multiple loci. In a quest to define the extent of variation in telomere length, we screened 112 wild-type Saccharomyces sensu stricto isolates. We found extensive telomere length variation in S. paradoxus isolates. This phenotype correlated with their geographic origin: European strains were observed to have extremely short telomeres (<150 bp), whereas American isolates had telomeres approximately three times as long (>400 bp). Insertions of a URA3 gene near telomeres allowed accurate analysis of individual telomere lengths and telomere position effect (TPE). Crossing the American and European strains resulted in F1 spores with a continuum of telomere lengths consistent with what would be predicted if many quantitative trait loci (QTLs) were involved in length maintenance. Variation in TPE is similarly quantitative but only weakly correlated with telomere length. Genotyping F1 segregants indicated several QTLs associated with telomere length and silencing variation. These QTLs include likely candidate genes but also map to regions where there are no known genes involved in telomeric properties. We detected transgressive segregation for both phenotypes. We validated by reciprocal hemizygosity that YKU80 and TLC1 are telomere-length QTLs in the two S. paradoxus subpopulations. Furthermore, we propose that sequence divergence within the Ku heterodimer generates negative epistasis within one of the allelic combinations (American-YKU70 and European-YKU80) resulting in very short telomeres.
PLoS Genetics 09/2009; 5(9):e1000659. · 8.69 Impact Factor
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ABSTRACT: The yKu protein of Saccharomyces cerevisiae is important for genome stability by repressing recombination involving telomeric sequences. The mechanism of this repression is not known, but silent heterochromatin such as HML, HMR, and telomeres are compartmentalized at the nuclear periphery and yKu is proposed to interact with these regions and to play a role in telomeric silencing and tethering. We have utilized ChIP on chip, quantitative PCR, and quantitative recombination assays to analyze yKu binding and its effect on genome stability in wild-type and mutant backgrounds. Our data suggest that, although yKu binds to the TG1-3 repeats and other parts of the genome when needed, such as during nonhomologous end-joining, it specifically binds to core X sequences in addition to the mating-type loci, HML and HMR. Association with core X occurred in the absence of Sir proteins, and enhanced binding was observed at silenced ends compared to nonsilenced ends. In contrast, binding to HML and HMR was totally dependent on Sir2-4p and partially dependent on Sir1p with a stronger association at HML in both MATa and MATalpha strains. Using yku80 separation-of-function mutants, we show a direct correlation between core X binding and recombination rate. We believe our findings support our hypothesis that yKu and core X play a pivotal role in maintaining genome stability through nuclear architecture by mediating a defensive fold-back structure at yeast chromosome ends.
Genetics 09/2009; 183(2):453-67, 1SI-13SI. · 4.01 Impact Factor
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Proceedings of the National Academy of Sciences 08/2009; 106(30):12217-8. · 9.68 Impact Factor
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ABSTRACT: Background
Infantile fibrosarcoma (IF) is a rare soft tissue sarcoma that presents either at birth or in the first year of life. Complete surgical resection is usually curative but chemotherapy may shrink the tumor to facilitate complete resection. This report describes the histologic changes and outcomes in four patients with IF treated with chemotherapy and surgical resection.ProcedureA retrospective review was performed of patients treated between 2000 and 2007.ResultsAll four patients are alive with excellent functional outcomes. The patients were diagnosed from birth up to 7 months of age; three had lower extremity tumors and one had a neck tumor. All patients received vincristine, cyclophosphamide, and actinomycin; one patient also received ifosfamide and etoposide after tumor progression. One tumor, arising from the neck, had rapid shrinkage. Two lower extremity tumors had only modest changes in dimensions but upon resection, the tumor bed contained fibrous tissue with exaggerated small caliber vessels. The fourth infant developed metastatic lesions in the central nervous system, orbits, lungs, and kidney after complete removal of the primary tumor. The metastatic lesions responded to chemotherapy and have remained stable for over 3 years.ConclusionsIF is a chemosensitive tumor. In patients where a clinical response is not apparent, cytoreduction of the tumor and replacement with fibrotic and fibrovascular tissue may facilitate gross-total resection. The chemotherapy-responsiveness of this tumor may abrogate unfavorable features such as metastatic or residual tumor. Pediatr Blood Cancer 2009;53:23–27. © 2009 Wiley-Liss, Inc.
Pediatric Blood & Cancer 07/2009; 53(1):23 - 27. · 1.89 Impact Factor
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ABSTRACT: Infantile fibrosarcoma (IF) is a rare soft tissue sarcoma that presents either at birth or in the first year of life. Complete surgical resection is usually curative but chemotherapy may shrink the tumor to facilitate complete resection. This report describes the histologic changes and outcomes in four patients with IF treated with chemotherapy and surgical resection.
A retrospective review was performed of patients treated between 2000 and 2007.
All four patients are alive with excellent functional outcomes. The patients were diagnosed from birth up to 7 months of age; three had lower extremity tumors and one had a neck tumor. All patients received vincristine, cyclophosphamide, and actinomycin; one patient also received ifosfamide and etoposide after tumor progression. One tumor, arising from the neck, had rapid shrinkage. Two lower extremity tumors had only modest changes in dimensions but upon resection, the tumor bed contained fibrous tissue with exaggerated small caliber vessels. The fourth infant developed metastatic lesions in the central nervous system, orbits, lungs, and kidney after complete removal of the primary tumor. The metastatic lesions responded to chemotherapy and have remained stable for over 3 years.
IF is a chemosensitive tumor. In patients where a clinical response is not apparent, cytoreduction of the tumor and replacement with fibrotic and fibrovascular tissue may facilitate gross-total resection. The chemotherapy-responsiveness of this tumor may abrogate unfavorable features such as metastatic or residual tumor.
Pediatric Blood & Cancer 05/2009; 53(1):23-7. · 1.89 Impact Factor
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ABSTRACT: Acute and late changes in magnetic resonance imaging of the pediatric brain have been described after radiotherapy (RT). We report the post-RT neuroimaging changes in the posterior fossa after intensity-modulated RT (IMRT) in children with medulloblastoma and contrast them with those of leptomeningeal disease.
We performed a retrospective review of 53 consecutive children with medulloblastoma who were treated with craniospinal RT followed by IMRT to the posterior fossa and chemotherapy between 1997 and 2006.
After IMRT to the posterior fossa, 8 (15%) of 53 patients developed increased fluid-attenuated inversion-recovery signal changes in the brainstem or cerebellum and patchy, multifocal, nodular contrast enhancement at a median of 6 months. The enhancement superficially resembled leptomeningeal disease. However, the enhancement resolved without intervention at a median of 6 months later. The accompanying fluid-attenuated inversion-recovery signal changes occasionally preceded the enhancement, were often parenchymal in location, and resolved or persisted to a lesser degree. All 8 patients with transient magnetic resonance imaging changes in the posterior fossa were alive at last follow-up. In contrast, leptomeningeal disease occurred in 8 (15%) of our 53 patients at a median of 19.5 months after IMRT completion. Of these 8 patients, 7 demonstrated initial nodular enhancement outside the conformal field, and 7 patients died.
Magnetic resonance imaging changes can occur in the posterior fossa of children treated with IMRT for medulloblastoma. In our experience, these transient changes occur at a characteristic time and location after RT, allowing them to be distinguished from leptomeningeal disease.
International journal of radiation oncology, biology, physics 06/2008; 73(1):214-21. · 4.59 Impact Factor
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ABSTRACT: The Ku heterodimer, comprised of Ku70 and Ku80 subunits, is a conserved complex involved in nonhomologous end-joining (NHEJ). However, it also functions in maintenance of telomeres, chromosome termini normally resistant to end-joining events. To elucidate the spatial organization of these functions, we rationally guided Ku mutagenesis in yeast with real-valued evolutionary trace (rvET). This revealed two ancestrally related alpha-helices: one on the Ku70 surface that is required in yeast for NHEJ, and a second on the Ku80 surface that is required in yeast for telomeric heterochromatin formation. When bound to a DNA end, the surface containing the NHEJ-specific Ku70 helix is oriented toward the DNA terminus, whereas the surface containing the telomeric function-specific Ku80 helix faces inward, toward telomeric chromatin, when bound to a telomere. We propose a 'two-face' model for Ku and that divergent evolution of these faces allowed Ku's dual role in NHEJ and telomere maintenance.
Nature Structural & Molecular Biology 05/2007; 14(4):301-7. · 12.71 Impact Factor
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ABSTRACT: Telomeres, the natural termini of eukaryotic chromosomes, have been the subject of intense interest during the last decade because of the roles that these chromosome termini perform in both cancer and aging. As we become more cognizant of the consequences of telomere dysfunction on several aspects of human health, significant attention is focused on understanding at a molecular level how the many telomere-associated factors perform their activities.
Current Opinion in Cell Biology 07/2006; 18(3):247-53. · 12.90 Impact Factor
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ABSTRACT: Cartilage-hair hypoplasia (CHH), also known as metaphyseal chondrodysplasia McKusick type (OMIM no. 250250), is an autosomal recessive, multi-systemic disease characterized by disproportionate short stature, fine and sparse hair, deficient cellular immunity and a predisposition to malignancy. It is caused by mutations in RMRP, the RNA component of the ribonucleoprotein complex RNase MRP, and, thus, CHH represents one of few Mendelian disorders caused by mutations in a nuclear encoded, non-coding RNA. While studies in yeast indicate that RMRP contributes to diverse cellular functions, the pathogenesis of the human condition is unknown. Studies of our CHH patient cohort revealed mutations in both the promoter and the transcribed region of RMRP. While mutations in the promoter abolished transcription in vitro, RMRP RNA levels in patients with transcribed mutations were also decreased suggesting an unstable RNA. RMRP mutations introduced into the yeast ortholog, NME1, exhibited normal mitochondrial function, chromosomal segregation and cell cycle progression, while a CHH fibroblast cell line exhibited normal mitochondrial content. However, the most commonly found mutation in CHH patients, 70A>G, caused an alteration in ribosomal processing by altering the ratio of the short versus the long form of the 5.8S rRNA in yeast. Transcriptional profiling of CHH patient RNAs showed upregulation of several cytokines and cell cycle regulatory genes, one of which has been implicated in chondrocyte hypertrophy. These data suggest that alteration of ribosomal processing in CHH is associated with altered cytokine signalling and cell cycle progression in terminally differentiating cells in the lymphocytic and chondrocytic cell lineages.
Human Molecular Genetics 01/2006; 14(23):3723-40. · 7.64 Impact Factor
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ABSTRACT: Previous work in budding yeast has indicated that telomeres are protected, at least in part, from the action of Exo1, which degrades the C-rich strand of partially uncapped telomeres. To explore this further, we examined the consequences of Exo1-mediated activity in strains that lacked Ku, telomerase, or both. Loss of Exo1 partially rescued the telomere length defect in a yku80delta strain, demonstrating that exonuclease action can directly contribute to telomere shortening. The rapid loss of inviability displayed by a yku80delta est2delta strain was also partially alleviated by an exo1delta mutation, further supporting the proposal that Exo1 is one target of the activities that normally protect wild-type telomeres. Conversely, however, Exo1 activity was also capable of enhancing telomere function and consequently cell proliferation, by contributing to a telomerase-independent pathway for telomere maintenance. The recovery of recombination-dependent survivors that arose in a yku80delta est2delta strain was partially dependent on Exo1 activity. Furthermore, the types of recombination events that facilitate telomerase-independent survival were influenced by Exo1 activity, in both est2delta and yku80delta est2delta strains. These data demonstrate that Exo1 can make either positive or negative contributions to telomere function and cell viability, depending on whether telomerase or recombination is utilized to maintain telomere function.
Genetics 05/2004; 166(4):1651-9. · 4.01 Impact Factor
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ABSTRACT: The Ku heterodimer functions at two kinds of DNA ends: telomeres and double-strand breaks. The role that Ku plays at these two classes of termini must be distinct, because Ku is required for accurate and efficient joining of double-strand breaks while similar DNA repair events are normally prohibited at chromosome ends. Toward defining these functional differences, we have identified eight mutations in the large subunit of the Saccharomyces cerevisiae Ku heterodimer (YKU80) which retain the ability to repair double-strand breaks but are severely impaired for chromosome end protection. Detailed characterization of these mutations, referred to as yku80(tel) alleles, has revealed that Ku performs functionally distinct activities at subtelomeric chromatin versus the end of the chromosome, and these activities are separable from Ku's role in telomere length regulation. While at the chromosome terminus, we propose that Ku participates in two different activities: it facilitates telomerase-mediated G-strand synthesis, thereby contributing to telomere length regulation, and it separately protects against resection of the C-strand, thereby contributing to protection of chromosome termini. Furthermore, we propose that the Ku heterodimer performs discrete sets of functions at chromosome termini and at duplex subtelomeric chromatin, via separate interactions with these two locations. Based on homology modeling with the human Ku structure, five of the yku80(tel) alleles mutate residues that are conserved between the yeast and human Ku80 proteins, suggesting that these mutations probe activities that are shared between yeast and humans.
Molecular and Cellular Biology 12/2003; 23(22):8202-15. · 5.53 Impact Factor
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Genes & Development 11/2003; 17(19):2347-50. · 11.66 Impact Factor
