Pot1b deletion and telomerase haploinsufficiency in mice initiate an ATR-dependent DNA damage response and elicit phenotypes resembling dyskeratosis congenita

The Protection of telomeres 1 (POT1) protein is a single-stranded telomere binding protein that is essential for proper maintenance of telomere length. Disruption of POT1 function leads to chromosome instability and loss of cellular viability. Here, we show that targeted deletion of the mouse Pot1b gene results in increased apoptosis in highly proliferative tissues. In the setting of telomerase haploinsufficiency, loss of Pot1b results in depletion of germ cells and complete bone marrow failure due to increased apoptosis, culminating in premature death. Pot1b^−/− mTR^+/− hematopoietic progenitor and stem cells display markedly reduced survival potential in vitro. Accelerated telomere shortening, increased G overhang and elevated number of chromosome end-to-end fusions that initiate an ATR-dependent DNA damage response were also observed. These results indicate an essential role for Pot1b in the maintenance of genome integrity and the long-term viability of proliferative tissues in the setting of telomerase deficiency. Interestingly, these phenotypes closely resemble those found in the human disease dyskeratosis congenita (DC), an inherited syndrome characterized by bone marrow failure, hyperpigmentation, and nail dystrophy. We anticipate that this mouse will serve as a useful model to further understand the pathophysiology of DC.     

Defective Repair of Oxidative Base Lesions by the DNA Glycosylase Nth1 Associates with Multiple Telomere Defects

Telomeres are chromosome end structures and are essential for maintenance of genome stability. Highly repetitive telomere sequences appear to be susceptible to oxidative stress-induced damage. Oxidation may therefore have a severe impact on telomere integrity and function. A wide spectrum of oxidative pyrimidine-derivatives has been reported, including thymine glycol (Tg), that are primarily removed by a DNA glycosylase, Endonuclease III-like protein 1 (Nth1). Here, we investigate the effect of Nth1 deficiency on telomere integrity in mice. Nth1 null (Nth1^−/−) mouse tissues and primary MEFs harbor higher levels of Endonuclease III-sensitive DNA lesions at telomeric repeats, in comparison to a non-telomeric locus. Furthermore, oxidative DNA damage induced by acute exposure to an oxidant is repaired slowly at telomeres in Nth1^−/− MEFs. Although telomere length is not affected in the hematopoietic tissues of Nth1^−/− adult mice, telomeres suffer from attrition and increased recombination and DNA damage foci formation in Nth1^−/− bone marrow cells that are stimulated ex vivo in the presence of 20% oxygen. Nth1 deficiency also enhances telomere fragility in mice. Lastly, in a telomerase null background, Nth1^−/− bone marrow cells undergo severe telomere loss at some chromosome ends and cell apoptosis upon replicative stress. These results suggest that Nth1 plays an important role in telomere maintenance and base repair against oxidative stress-induced base modifications. The fact that telomerase deficiency can exacerbate telomere shortening in Nth1 deficient mouse cells supports that base excision repair cooperates with telomerase to maintain telomere integrity.     

Utilizing Murine Inducible Telomerase Alleles in the Studies of Tissue Degeneration/Regeneration and Cancer

Telomere dysfunction-induced loss of genome integrity and its associated DNA damage signaling and checkpoint responses are well-established drivers that cause tissue degeneration during ageing. Cancer, with incidence rates greatly increasing with age, is characterized by short telomere lengths and high telomerase activity. To study the roles of telomere dysfunction and telomerase reactivation in ageing and cancer, the protocol shows how to generate two murine inducible telomerase knock-in alleles 4-Hydroxytamoxifen (4-OHT)-inducible TERT-Estrogen Receptor (mTERT-ER) and Lox-Stopper-LoxTERT (LSL-mTERT). The protocol describes the procedures to induce telomere dysfunction and reactivate telomerase activity in mTERT-ER and LSL-mTERT mice in vivo. The representative data show that reactivation of telomerase activity can ameliorate the tissue degenerative phenotypes induced by telomere dysfunction. In order to determine the impact of telomerase reactivation on tumorigenesis, we generated prostate tumor model G4 PB-Cre4 Pten^L/L p53^L/L LSL-mTERT^L/L and thymic T-cell lymphoma model G4 Atm^-/- mTERT^ER/ER. The representative data show that telomerase reactivation in the backdrop of genomic instability induced by telomere dysfunction can greatly enhance tumorigenesis. The protocol also describes the procedures used to isolate neural stem cells (NSCs) from mTERT-ER and LSL-mTERT mice and reactivate telomerase activity in NSCs in vitro. The representative data show that reactivation of telomerase can enhance the self-renewal capability and neurogenesis in vitro. Finally, the protocol describes the procedures for performing telomere FISH (Fluorescence In Situ Hybridization) on both mouse FFPE (Formalin Fixed and Paraffin Embedded) brain tissues and metaphase chromosomes of cultured cells.     

Short and dysfunctional telomeres protect from allergen‐induced airway inflammation

Asthma is a chronic inflammatory disease affecting 300 million people worldwide. As telomere shortening is a well‐established hallmark of aging and that asthma incidence decreases with age, here we aimed to study the role of short telomeres in asthma pathobiology. To this end, wild‐type and telomerase‐deficient mice with short telomeres (third‐generation (G3 Tert ^−/− mice)) were challenged with intranasal house dust mite (HDM) extract. We also challenged with HDM wild‐type mice in which we induced a telomere dysfunction by the administration of 6‐thio‐2´‐deoxyguanosine (6‐thio‐dG). Following HDM exposure, G3 Tert ^−/− and 6‐thio‐dG treated mice exhibited attenuated eosinophil counts and presence of hematopoietic stem cells in the bone marrow, as well as lower levels of IgE and circulating eosinophils. Accordingly, both G3 Tert ^−/− and 6‐thio‐dG treated wild‐type mice displayed reduced airway hyperresponsiveness (AHR), as indicated by decreased airway remodeling and allergic airway inflammation markers in the lung. Furthermore, G3 Tert ^−/− and 6‐thio‐dG treated mice showed lower differentiation of Club cells, attenuating goblet cell hyperplasia. Club cells of G3 Tert ^−/− and 6‐thio‐dG treated mice displayed increased DNA damage and senescence and reduced proliferation. Thus, short/dysfunctional telomeres play a protective role in murine asthma by impeding both AHR and mucus secretion after HDM exposure. Therefore, our findings imply that telomeres play a relevant role in allergen‐induced airway inflammation.     

Break-Induced Replication Requires DNA Damage-Induced Phosphorylation of Pif1 and Leads to Telomere Lengthening

Broken replication forks result in DNA breaks that are normally repaired via homologous recombination or break induced replication (BIR). Mild insufficiency in the replicative ligase Cdc9 in budding yeast Saccharomyces cerevisiae resulted in a population of cells with persistent DNA damage, most likely due to broken replication forks, constitutive activation of the DNA damage checkpoint and longer telomeres. This telomere lengthening required functional telomerase, the core DNA damage signaling cascade Mec1-Rad9-Rad53, and the components of the BIR repair pathway – Rad51, Rad52, Pol32, and Pif1. The Mec1-Rad53 induced phosphorylation of Pif1, previously found necessary for inhibition of telomerase at double strand breaks, was also important for the role of Pif1 in BIR and telomere elongation in cdc9-1 cells. Two other mutants with impaired DNA replication, cdc44-5 and rrm3Δ, were similar to cdc9-1: their long telomere phenotype was dependent on the Pif1 phosphorylation locus. We propose a model whereby the passage of BIR forks through telomeres promotes telomerase activity and leads to telomere lengthening.     

Short Telomeres Initiate Telomere Recombination in Primary and Tumor Cells

Human tumors that lack telomerase maintain telomeres by alternative lengthening mechanisms. Tumors can also form in telomerase-deficient mice; however, the genetic mechanism responsible for tumor growth without telomerase is unknown. In yeast, several different recombination pathways maintain telomeres in the absence of telomerase—some result in telomere maintenance with minimal effects on telomere length. To examine non-telomerase mechanisms for telomere maintenance in mammalian cells, we used primary cells and lymphomas from telomerase-deficient mice (mTR−/− and Eμmyc+mTR−/−) and CAST/EiJ mouse embryonic fibroblast cells. These cells were analyzed using pq-ratio analysis, telomere length distribution outliers, CO-FISH, Q-FISH, and multicolor FISH to detect subtelomeric recombination. Telomere length was maintained during long-term growth in vivo and in vitro. Long telomeres, characteristic of human ALT cells, were not observed in either late passage or mTR−/− tumor cells; instead, we observed only minimal changes in telomere length. Telomere length variation and subtelomeric recombination were frequent in cells with short telomeres, indicating that length maintenance is due to telomeric recombination. We also detected telomere length changes in primary mTR−/− cells that had short telomeres. Using mouse mTR+/− and human hTERT+/− primary cells with short telomeres, we found frequent length changes indicative of recombination. We conclude that telomere maintenance by non-telomerase mechanisms, including recombination, occurs in primary cells and is initiated by short telomeres, even in the presence of telomerase. Most intriguing, our data indicate that some non-telomerase telomere maintenance mechanisms occur without a significant increase in telomere length.     

The Mutyh Base Excision Repair Gene Influences the Inflammatory Response in a Mouse Model of Ulcerative Colitis

Background

The Mutyh DNA glycosylase is involved in the repair of oxidized DNA bases. Mutations in the human MUTYH gene are responsible for colorectal cancer in familial adenomatous polyposis. Since defective DNA repair genes might contribute to the increased cancer risk associated with inflammatory bowel diseases, we compared the inflammatory response of wild-type and Mutyh^−/− mice to oxidative stress.

Methodology/Principal Findings

The severity of colitis, changes in expression of genes involved in DNA repair and inflammation, DNA 8-oxoguanine levels and microsatellite instability were analysed in colon of mice treated with dextran sulfate sodium (DSS). The Mutyh^−/− phenotpe was associated with a significant accumulation of 8-oxoguanine in colon DNA of treated mice. A single DSS cycle induced severe acute ulcerative colitis in wild-type mice, whereas lesions were modest in Mutyh^−/− mice, and this was associated with moderate variations in the expression of several cytokines. Eight DSS cycles caused chronic colitis in both wild-type and Mutyh^−/− mice. Lymphoid hyperplasia and a significant reduction in Foxp3⁺ regulatory T cells were observed only in Mutyh^−/− mice.

Conclusions

The findings indicate that, in this model of ulcerative colitis, Mutyh plays a major role in maintaining intestinal integrity by affecting the inflammatory response.     

A genetic interaction between RAP1 and telomerase reveals an unanticipated role for RAP1 in telomere maintenance

RAP1 is one of the components of shelterin, the capping complex at chromosome ends or telomeres, although its role in telomere length maintenance and protection has remained elusive. RAP1 also binds subtelomeric repeats and along chromosome arms, where it regulates gene expression and has been shown to function in metabolism control. Telomerase is the enzyme that elongates telomeres, and its deficiency causes a premature aging in humans and mice. We describe an unanticipated genetic interaction between RAP1 and telomerase. While RAP1 deficiency alone does not impact on mouse survival, mice lacking both RAP1 and telomerase show a progressively decreased survival with increasing mouse generations compared to telomerase single mutants. Telomere shortening is more pronounced in Rap1^?/? Terc^?/? doubly deficient mice than in the single‐mutant Terc^?/? counterparts, leading to an earlier onset of telomere‐induced DNA damage and degenerative pathologies. Telomerase deficiency abolishes obesity and liver steatohepatitis provoked by RAP1 deficiency. Using genomewide ChIP sequencing, we find that progressive telomere shortening owing to telomerase deficiency leads to re‐localization of RAP1 from telomeres and subtelomeric regions to extratelomeric sites in a genomewide manner. These findings suggest that although in the presence of sufficient telomere reserve RAP1 is not a key factor for telomere maintenance and protection, it plays a crucial role in the context of telomerase deficiency, thus in agreement with its evolutionary conservation as a telomere component from yeast to humans.     

Genomic instability in both wild-type and telomerase null MEFs

To examine chromosome instability in the absence of telomerase, we established mouse embryonic fibroblast (MEF) lines from late generation mTR–/– and wild-type animals and examined metaphases using telomere fluorescence in situ hybridization (FISH) and spectral karyotyping (SKY). In early passages, mTR–/– G6 cell lines showed more chromosome ends with no telomere signal, more chromosome end-to-end fusions and greater radiosensitivity than wild-type lines. At later passages, however, the rate of genomic instability in the wild-type MEFs increased to a level similar or higher than seen in the mTR–/– G6 cell lines. This high degree of instability in wild-type MEF lines suggests that post-crisis MEFs should not be considered genetically defined cell lines. Surprisingly, the increased radiosensitivity seen in early passage mTR–/– G6 cultures was lost after crisis. Both post-crisis mTR–/– G6 MEFs and wild-type MEFs showed loss of p53 and -H2AX phosphorylation in response to irradiation, indicating a loss of DNA damage checkpoints.Electronic Supplementary Material Supplementary material is available for this article at .     

TAp73 is required for macrophage-mediated innate immunity and the resolution of inflammatory responses

The multiple isoforms of p73, a member of the p53 family, share the ability to modulate p53 activities but also have unique properties, leading to a complex and poorly understood functional network. In vivo, p73 isoforms have been implicated in tumor suppression (TAp73^−/− mice), DNA damage (ΔNp73^−/− mice) and development (p73^−/− mice). In this study, we investigated whether TAp73 contributes to innate immunity and septic shock. In response to a lethal lipopolysaccharide (LPS) challenge, TAp73^−/− mice showed higher blood levels of proinflammatory cytokines and greater mortality than their wild-type littermates. In vitro, TAp73^−/− macrophages exhibited elevated production of tumor necrosis factor alpha , interleukin-6 and macrophage inflammatory protein-2 as well as prolonged survival, decreased phagocytosis and increased major histocompatibility complex class II expression. Mice depleted of endogenous macrophages and reconstituted with TAp73^−/− macrophages showed increased sensitivity to LPS challenge. These results suggest that macrophage polarization is altered in the absence of TAp73 such that maintenance of the M1 effector phenotype is prolonged at the expense of the M2 phenotype, thus impairing resolution of the inflammatory response. Our data indicate that TAp73 has a role in macrophage polarization and innate immunity, enhancing the action field of this important regulatory molecule.     

C/EBP homologous protein deficiency enhances hematopoietic stem cell function via reducing ATF3/ROS‐induced cell apoptosis

Hematopoietic stem cells (HSCs) reside in a quiescent niche to reserve their capacity of self‐renewal. Upon hematopoietic injuries, HSCs enter the cell cycle and encounter protein homeostasis problems caused by accumulation of misfolded proteins. However, the mechanism by which protein homeostasis influences HSC function and maintenance remains poorly understood. Here, we show that C/EBP homologous protein (CHOP), demonstrated previously to induces cell death upon unfolded protein response (UPR), plays an important role in HSCs regeneration. CHOP^−/− mice showed normal hematopoietic stem and progenitor cell frequencies in steady state. However, when treated with 5‐FU, CHOP deficiency resulted in higher survival rates, associated with an increased number of HSCs and reduced level of apoptosis. In serial competitive transplantation experiments, CHOP^−/− HSCs showed a dramatic enhancement of repopulation ability and a reduction of protein aggresomes. Mechanistically, CHOP deletion causes reduced ATF3 expression and further leads to decreased protein aggregation and ROS. In addition, CHOP^−/− HSCs exhibited an increased resistance to IR‐induced DNA damage and improved HSCs homeostasis and function in telomere dysfunctional (G3Terc ^−/−) mice. In summary, these findings disclose a new role of CHOP in the regulation of the HSCs function and homeostasis through reducing ATF3 and ROS signaling.     

Small, Acid-Soluble Spore Proteins of the α/β Type Do Not Protect the DNA in Bacillus subtilis Spores against Base Alkylation

Ethyl methanesulfonate (EMS) killed wild-type Bacillus subtilis spores as rapidly as spores lacking small, acid-soluble proteins (SASP) of the α/β type (α⁻β⁻ spores), and 20% of the survivors had obvious mutations. A recA mutation increased the EMS sensitivity of wild-type and α⁻β⁻ spores similarly but reduced their mutagenesis; EMS treatment of dormant spores also resulted in the induction of RecA synthesis during spore germination. EMS generated similar levels of alkylated bases in wild-type and α⁻β⁻ spore DNAs, in purified DNA, or in DNA saturated with α/β-type SASP. Ethylene oxide (EtO) also generated similar levels of base alkylation in wild-type and α⁻β⁻ spore DNAs. These data indicate that EMS and EtO kill spores at least in part by DNA damage but that α/β-type SASP, which protect DNA against many types of damage, do not protect spore DNA from base alkylation.     

Synergistic Interaction of Rnf8 and p53 in the Protection against Genomic Instability and Tumorigenesis

Rnf8 is an E3 ubiquitin ligase that plays a key role in the DNA damage response as well as in the maintenance of telomeres and chromatin remodeling. Rnf8^−/− mice exhibit developmental defects and increased susceptibility to tumorigenesis. We observed that levels of p53, a central regulator of the cellular response to DNA damage, increased in Rnf8^−/− mice in a tissue- and cell type–specific manner. To investigate the role of the p53-pathway inactivation on the phenotype observed in Rnf8^−/− mice, we have generated Rnf8^−/−p53^−/− mice. Double-knockout mice showed similar growth retardation defects and impaired class switch recombination compared to Rnf8^−/− mice. In contrast, loss of p53 fully rescued the increased apoptosis and reduced number of thymocytes and splenocytes in Rnf8^−/− mice. Similarly, the senescence phenotype of Rnf8^−/− mouse embryonic fibroblasts was rescued in p53 null background. Rnf8^−/−p53^−/− cells displayed defective cell cycle checkpoints and DNA double-strand break repair. In addition, Rnf8^−/−p53^−/− mice had increased levels of genomic instability and a remarkably elevated tumor incidence compared to either Rnf8^−/− or p53^−/− mice. Altogether, the data in this study highlight the importance of p53-pathway activation upon loss of Rnf8, suggesting that Rnf8 and p53 functionally interact to protect against genomic instability and tumorigenesis.