Telomere synthesis in cancer cells and stem cells involves trafficking of telomerase to Cajal bodies, and telomerase is thought to be recruited to telomeres through interactions with telomere-binding ...proteins. Here, we show that the OB-fold domain of the telomere-binding protein TPP1 recruits telomerase to telomeres through an association with the telomerase reverse transcriptase TERT. When tethered away from telomeres and other telomere-binding proteins, the TPP1 OB-fold domain is sufficient to recruit telomerase to a heterologous chromatin locus. Expression of a minimal TPP1 OB-fold inhibits telomere maintenance by blocking access of telomerase to its cognate binding site at telomeres. We identify amino acids required for the TPP1-telomerase interaction, including specific loop residues within the TPP1 OB-fold domain and individual residues within TERT, some of which are mutated in a subset of pulmonary fibrosis patients. These data define a potential interface for telomerase-TPP1 interaction required for telomere maintenance and implicate defective telomerase recruitment in telomerase-related disease.
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► Enforced expression of telomerase forms neo-Cajal bodies at telomeres ► TPP1 OB-fold domain recruits telomerase to a heterologous chromatin locus ► TPP1-OB alone sequesters telomerase within Cajal bodies and causes telomere shortening ► OB-fold mutations and some disease mutations in TERT block telomerase recruitment
The shelterin component TPP1 recruits telomerase from storage in Cajal bodies via a direct interaction with the enzyme’s catalytic subunit TERT. Mutations in TERT found associated with an idiopathic lung disease disrupt the interaction and impair telomerase mobilization.
Telomere biology disorders (TBDs) are a group of rare diseases caused by mutations that impair telomere maintenance. Mutations that cause reduced levels of TERC/hTR, the telomerase RNA component, are ...found in most TBD patients and include loss-of-function mutations in hTR itself, in hTR-binding proteins NOP10, NHP2, NAF1, ZCCHC8, and dyskerin (DKC1), and in proteins required for hTR processing (PARN). These patients show diverse clinical presentations that most commonly include bone marrow failure (BMF)/aplastic anemia (AA), pulmonary fibrosis, and liver cirrhosis. There are no curative therapies for TBD patients. An understanding of hTR biogenesis, maturation, and degradation has identified pathways and pharmacological agents targeting the poly(A) polymerase PAPD5, which adds 3′-oligoadenosine tails to hTR to promote hTR degradation, and TGS1, which modifies the 5′-cap structure of hTR to enhance degradation, as possible therapeutic approaches. Critical next steps will be clinical trials to establish the effectiveness and potential side effects of these compounds in TBD patients.
Mutations in genes associated with telomere maintenance occur in telomere biology disorders (TBDs). These poorly treated disorders are characterized by multisystemic phenotypes including bone marrow failure/aplastic anemia, pulmonary fibrosis, liver cirrhosis, and increased cancer incidence.Mutations that cause reduced levels of TERC/hTR, the telomerase RNA component, are found in most TBD patients and include loss-of-function mutations in hTR itself, in several hTR-binding proteins, and in proteins required for post-transcriptional hTR processing. These mutations culminate in increased hTR 3′-end oligo-adenylation by the noncanonical poly(A) polymerase PAPD5, which triggers exonucleolytic decay of the telomerase RNA component.Recent evidence shows that genetic or chemical inhibition of PAPD5 can rescue telomere homeostasis and cellular functionality in both murine and human models of TBDs, indicating this could serve as a possible therapeutic approach for several TBDs.
Aging is a major risk factor in many forms of late-onset neurodegenerative disorders. The ability to recapitulate age-related characteristics of human neurons in culture will offer unprecedented ...opportunities to study the biological processes underlying neuronal aging. Here, we show that using a recently demonstrated microRNA-based cellular reprogramming approach, human fibroblasts from postnatal to near centenarian donors can be efficiently converted into neurons that maintain multiple age-associated signatures. Application of an epigenetic biomarker of aging (referred to as epigenetic clock) to DNA methylation data revealed that the epigenetic ages of fibroblasts were highly correlated with corresponding age estimates of reprogrammed neurons. Transcriptome and microRNA profiles reveal genes differentially expressed between young and old neurons. Further analyses of oxidative stress, DNA damage and telomere length exhibit the retention of age-associated cellular properties in converted neurons from corresponding fibroblasts. Our results collectively demonstrate the maintenance of age after neuronal conversion.
The differentiation of patient-derived induced pluripotent stem cells (iPSCs) to committed fates such as neurons, muscle and liver is a powerful approach for understanding key parameters of human ...development and disease. Whether undifferentiated iPSCs themselves can be used to probe disease mechanisms is uncertain. Dyskeratosis congenita is characterized by defective maintenance of blood, pulmonary tissue and epidermal tissues and is caused by mutations in genes controlling telomere homeostasis. Short telomeres, a hallmark of dyskeratosis congenita, impair tissue stem cell function in mouse models, indicating that a tissue stem cell defect may underlie the pathophysiology of dyskeratosis congenita. Here we show that even in the undifferentiated state, iPSCs from dyskeratosis congenita patients harbour the precise biochemical defects characteristic of each form of the disease and that the magnitude of the telomere maintenance defect in iPSCs correlates with clinical severity. In iPSCs from patients with heterozygous mutations in TERT, the telomerase reverse transcriptase, a 50% reduction in telomerase levels blunts the natural telomere elongation that accompanies reprogramming. In contrast, mutation of dyskerin (DKC1) in X-linked dyskeratosis congenita severely impairs telomerase activity by blocking telomerase assembly and disrupts telomere elongation during reprogramming. In iPSCs from a form of dyskeratosis congenita caused by mutations in TCAB1 (also known as WRAP53), telomerase catalytic activity is unperturbed, yet the ability of telomerase to lengthen telomeres is abrogated, because telomerase mislocalizes from Cajal bodies to nucleoli within the iPSCs. Extended culture of DKC1-mutant iPSCs leads to progressive telomere shortening and eventual loss of self-renewal, indicating that a similar process occurs in tissue stem cells in dyskeratosis congenita patients. These findings in iPSCs from dyskeratosis congenita patients reveal that undifferentiated iPSCs accurately recapitulate features of a human stem cell disease and may serve as a cell-culture-based system for the development of targeted therapeutics.
Acute treatment with replication-stalling chemotherapeutics causes reversal of replication forks. BRCA proteins protect reversed forks from nucleolytic degradation, and their loss leads to ...chemosensitivity. Here, we show that fork degradation is no longer detectable in BRCA1-deficient cancer cells exposed to multiple cisplatin doses, mimicking a clinical treatment regimen. This effect depends on increased expression and chromatin loading of PRIMPOL and is regulated by ATR activity. Electron microscopy and single-molecule DNA fiber analyses reveal that PRIMPOL rescues fork degradation by reinitiating DNA synthesis past DNA lesions. PRIMPOL repriming leads to accumulation of ssDNA gaps while suppressing fork reversal. We propose that cells adapt to repeated cisplatin doses by activating PRIMPOL repriming under conditions that would otherwise promote pathological reversed fork degradation. This effect is generalizable to other conditions of impaired fork reversal (e.g., SMARCAL1 loss or PARP inhibition) and suggests a new strategy to modulate cisplatin chemosensitivity by targeting the PRIMPOL pathway.
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•Multiple cisplatin doses suppress reversed fork degradation in BRCA-deficient cells•The PRIMPOL adaptive response suppresses fork reversal and leads to ssDNA gaps•The ATR kinase regulates the PRIMPOL-mediated adaptive response•Impaired fork reversal shifts the balance toward PRIMPOL-mediated repriming
Reversed replication forks are degraded in BRCA-deficient cells. Quinet et al. show that treatment with multiple cisplatin doses promotes PRIMPOL repriming while suppressing fork reversal and preventing degradation. These studies identify a new function of PRIMPOL in the replication stress response and explain how cells adapt to multiple drug doses.
The combination of induced pluripotent stem cell (iPSC) technology and targeted gene modification by homologous recombination (HR) represents a promising new approach to generate genetically ...corrected, patient‐derived cells that could be used for autologous transplantation therapies. This strategy has several potential advantages over conventional gene therapy including eliminating the need for immunosuppression, avoiding the risk of insertional mutagenesis by therapeutic vectors, and maintaining expression of the corrected gene by endogenous control elements rather than a constitutive promoter. However, gene targeting in human pluripotent cells has remained challenging and inefficient. Recently, engineered zinc finger nucleases (ZFNs) have been shown to substantially increase HR frequencies in human iPSCs, raising the prospect of using this technology to correct disease causing mutations. Here, we describe the generation of iPSC lines from sickle cell anemia patients and in situ correction of the disease causing mutation using three ZFN pairs made by the publicly available oligomerized pool engineering method (OPEN). Gene‐corrected cells retained full pluripotency and a normal karyotype following removal of reprogramming factor and drug‐resistance genes. By testing various conditions, we also demonstrated that HR events in human iPSCs can occur as far as 82 bps from a ZFN‐induced break. Our approach delineates a roadmap for using ZFNs made by an open‐source method to achieve efficient, transgene‐free correction of monogenic disease mutations in patient‐derived iPSCs. Our results provide an important proof of principle that ZFNs can be used to produce gene‐corrected human iPSCs that could be used for therapeutic applications. STEM CELLS 2011;29:1717–1726
Mutations in the retinoblastoma tumor suppressor gene Rb are involved in many forms of human cancer. In this study, we investigated the early consequences of inactivating Rb in the context of ...cellular reprogramming. We found that Rb inactivation promotes the reprogramming of differentiated cells to a pluripotent state. Unexpectedly, this effect is cell cycle independent, and instead reflects direct binding of Rb to pluripotency genes, including Sox2 and Oct4, which leads to a repressed chromatin state. More broadly, this regulation of pluripotency networks and Sox2 in particular is critical for the initiation of tumors upon loss of Rb in mice. These studies therefore identify Rb as a global transcriptional repressor of pluripotency networks, providing a molecular basis for previous reports about its involvement in cell fate pliability, and implicate misregulation of pluripotency factors such as Sox2 in tumorigenesis related to loss of Rb function.
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•The Rb tumor suppressor inhibits reprogramming of fibroblasts to iPSCs•The effect of Rb on reprogramming is independent of cell-cycle regulation•Rb promotes assembly of repressive chromatin at pluripotency network genes•Deletion of Sox2 prevents cancer initiation upon loss of Rb in mice
To investigate how the Rb tumor suppressor inhibits cellular dedifferentiation, Kareta et al. utilized iPSC reprogramming as a cellular system and observed that Rb restricts reprogramming by silencing pluripotency genes and networks. Rb repression of one pluripotency factor, Sox2, in particular, is critical to block cancer initiation in mice.
It is well established that short telomeres activate an ATM-driven DNA damage response that leads to senescence in terminally differentiated cells. However, technical limitations have hampered our ...understanding of how telomere shortening is signaled in human stem cells. Here, we show that telomere attrition induces ssDNA accumulation (G-strand) at telomeres in human pluripotent stem cells (hPSCs), but not in their differentiated progeny. This led to a unique role for ATR in the response of hPSCs to telomere shortening that culminated in an extended S/G2 cell cycle phase and a longer period of mitosis, which was associated with aneuploidy and mitotic catastrophe. Loss of p53 increased resistance to death, at the expense of increased mitotic abnormalities in hPSCs. Taken together, our data reveal an unexpected dominant role of ATR in hPSCs, combined with unique cell cycle abnormalities and, ultimately, consequences distinct from those observed in their isogenic differentiated counterparts.
Glioblastoma multiforme is the most severe form of brain cancer. First line therapy includes the methylating agent temozolomide and/or the chloroethylating nitrosoureas ...1-(2-chloroethyl)-1-nitrosourea; CNU nimustine 1-(4-amino-2-methyl-5-pyrimidinyl)methyl-3-(2-chloroethyl)-3-nitrosourea; ACNU, carmustine 1,3-bis(2-chloroethyl)-1-nitrosourea; BCNU, or lomustine 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea; CCNU. The mechanism of cell death after CNU treatment is largely unknown. Here we show that ACNU and BCNU induce apoptosis in U87MG p53 wild-type (p53wt) and U138MG p53 mutant (p53mt) glioma cells. However, contrary to what we observed previously for temozolomide, chloroethylating drugs are more toxic for p53-mutated glioma cells and induce both apoptosis and necrosis. Inactivation of p53 by pifithrin-alpha or siRNA down-regulation sensitized p53wt but not p53mt glioma cells to ACNU and BCNU. ACNU and BCNU provoke the formation of DNA double-strand breaks (DSB) in glioma cells that precede the onset of apoptosis and necrosis. Although these DSBs are repaired in p53wt cells, they accumulate in p53mt cells. Therefore, functional p53 seems to stimulate the repair of CNU-induced cross-links and/or DSBs generated from CNU-induced lesions. Expression analysis revealed an up-regulation of xpc and ddb2 mRNA in response to ACNU in U87MG but not U138MG cells, indicating p53 regulates a pathway that involves these DNA repair proteins. ACNU-induced apoptosis in p53wt glioma cells is executed via both the extrinsic and intrinsic apoptotic pathway, whereas in p53mt glioma cells, the mitochondrial pathway becomes activated. The data suggest that p53 has opposing effects in gliomas treated with methylating or chloroethylating agents and, therefore, the p53 status should be taken into account when deciding which therapeutic drug to use.
Highlights • DC is a genetic disease with features of stem cell dysfunction in diverse tissues. • Mutations in the telomere homeostasis genes underlie DC. • Diverse biochemical defects lead to ...impaired telomere maintenance in DC iPS cells. • DC iPS cells allow genotype–phenotype comparisons. • Severity of biochemical defects in DC iPS cells predicts disease severity.
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