53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\53BP1 deficiency combined with telomere dysfunction activates ATR-dependent DNA damage response.pdf" Abstract TRF1 protects mammalian telomeres from fusion and fragility. Depletion of TRF1 leads to telomere fusions as well as accumulation of ?-H2AX foci and activation of both the ataxia telangiectasia mutated (ATM)– and the ataxia telangiectasia and Rad3 related (ATR)–mediated deoxyribonucleic acid (DNA) damage response (DDR) pathways. 53BP1, which is also present at dysfunctional telomeres, is a target of ATM that accumulates at DNA double-strand breaks and favors nonhomologous end-joining (NHEJ) repair over ATM-dependent resection and homology-directed repair (homologous recombination [HR]). To address the role of 53BP1 at dysfunctional telomeres, we generated mice lacking TRF1 and 53BP1. 53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibroblasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. Concomitantly, double-mutant MEFs showed evidence of hyperactivation of the ATR-dependent DDR. In intact mice, combined 53BP1/TRF1 deficiency in stratified epithelia resulted in earlier onset of DNA damage and increased CHK1 phosphorylation during embryonic development, leading to aggravation of skin phenotypes. Some notes -CNIO paper -Maria Blasco corresponding author -The Journal of Cell Biology Paper -we generated mice lacking TRF1 and 53BP1 -53BP1 deficiency significantly rescued telomere fusions in mouse embryonic fibro - blasts (MEFs) lacking TRF1, but they showed evidence of a switch from the NHEJ- to HR-mediated repair of uncapped telomeres. --NHEJ: nonhomologous end-joining --HR: homologous recombination -53BP1 deficiency does not rescue growth defects of TRF1 -deleted MEFs -53BP1 deficiency rescues chromosome- type end to end fusions but results in increased telomere recombination associated with TRF1 abrogation -53BP1 deficiency in TRF1 -null MEFs leads to stronger CHK1 activation and telomeric RPA foci -Aggravation of TRF1 loss–mediated in vivo pathologies by 53BP1 deficiency -TRF1 ?/ ? K5-Cre 53BP1 ?/ ? mice show earlier onset of DNA damage and of DDR activation -Massive arrest of TRF1 ?/ ? K5-Cre 53BP1 ?/ ? keratinocytes in G2 -Longer telomeres in TRF1 ?/ ? K5-Cre 53BP1 ?/ ? mouse epidermis -Our work provides the first evidence of deleterious genetic interaction between a shelterin component and a DNA damage repair component, TRF1 and 53BP1, respectively. -knockout mice -immunoblotting -retroviral infections -immunofluorescence -CO-FISH -TRF analysis -histopathology -immunohistochemistry -FACS -Q-FISHA metabolic signature predicts biological age in mice "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-08-2014d0943\A metabolic signature predicts biological age in mice.pdf" Abstract Our understanding of the mechanisms by which aging is produced is still very limited. Here, we have determined the sera metabolite profile of 117 wild-type mice of different genetic backgrounds ranging from 8 to 129 weeks of age. This has allowed us to define a robust metabolomic signature and a derived metabolomic score that reliably/accurately predicts the age of wild-type mice. In the case of telomerase-deficient mice, which have a shortened lifespan, their metabolomic score predicts older ages than expected. Conversely, in the case of mice that overexpress telomerase, their metabolic score corresponded to younger ages than expected. Importantly, telomerase reactivation late in life by using a TERT-based gene therapy recently described by us significantly reverted the metabolic profile of old mice to that of younger mice, further confirming an anti-aging role for telomerase. Thus, the metabolomic signature associated with natural mouse aging accurately predicts aging produced by telomere shortening, suggesting that natural mouse aging is in part produced by presence of short telomeres. These results indicate that the metabolomic signature is associated with the biological age rather than with the chronological age. This constitutes one of the first aging-associated metabolomic signatures in a mammalian organism. Some Notes -Tomas-Loba, Bernardes de Jesus, and Maria Blasco paper -CNIO paper -Aging Cell paper -Maria Blasco is corresponding author -q de?ne a robust metabolomic signature and a derived metabolomic score that reliably/accurately predicts the age of wild-type mice -TERT-based gene therapy recently described by us signi?cantly reverted the metabolic pro?le of old mice to that of younger mice -there is a table of serum metabolic biomarkers -q The age biomarkers identi?ed here include lipids and other small molecules such as creatine, methionine, and uric acid. Whether these biomarkers play a role in aging or are due to secondary phenomena will require further investigation -q strong evidence that telomere loss signi?cantly contributes to natural mouse aging. -q estimate the impact of genetic modi?cations or treatments on mouse aging. -UPLC-MS . . .MS -data processed with MarkerLynx for MassLynx -multivariate model with SIMCA - P+ software -ChemSpider database -Kegg, Human Metabolome database, and lipid mapsA p53-dependent response limits epidermal stem cell functionality and organismal size in mice with short telomeres location of paper -"C:\Users\kurtw_000\Box Sync\DocDR\2014\01-24-2014d0943\A p53-dependent response limits epidermal stem cell functionality and organismal size in mice with short telomeres.pdf" notes and highlights -Together, these findings indicate the existence of a p53-dependent senescence response acting on stem/progenitor cells with dysfunctional telomeres that is actively limiting their contribution to tissue regeneration, thereby impinging on tissue fitness. -generating doubly deficient mice for telomerase and p53, Terc -/ - p53 -/- -p53 abrogation rescues epidermal and hair growth defects in telomerase-deficient mice with short telomeres -These findings argue that critically short telomeres can activate p53 during mouse development, which in turn limits the net expansion of tissues. (results are kind of surprising) -"Interestingly, given the fact that p53 deletion in mice with short telomeres eventually leads to accelerated tumor formation, the premature nature of the p53-mediated blockage on stem cell activation in these mice could serve as an ahead-of- time crucial first barrier against tumor progression. Alternatively, . . ." -"p53 abrogation could conceivably restitute stem cells functionality in other organs" -performed punch biopsiesA p53-mediated DNA damage response limits reprogramming to ensure iPS cell genomic integrity "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-31-2014d1002\Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells.pdf" The reprogramming of differentiated cells to pluripotent cells (induced pluripotent stem (iPS) cells) is known to be an inefficient process. We recently reported that cells with short telomeres cannot be reprogrammed to iPS cells despite their normal proliferation rates1, 2, probably reflecting the existence of ‘reprogramming barriers’ that abort the reprogramming of cells with uncapped telomeres. Here we show that p53 (also known as Trp53 in mice and TP53 in humans) is critically involved in preventing the reprogramming of cells carrying various types of DNA damage, including short telomeres, DNA repair deficiencies, or exogenously inflicted DNA damage. Reprogramming in the presence of pre-existing, but tolerated, DNA damage is aborted by the activation of a DNA damage response and p53-dependent apoptosis. Abrogation of p53 allows efficient reprogramming in the face of DNA damage and the generation of iPS cells carrying persistent DNA damage and chromosomal aberrations. These observations indicate that during reprogramming cells increase their intolerance to different types of DNA damage and that p53 is critical in preventing the generation of human and mouse pluripotent cells from suboptimal parental cells. some notes -q Reprogramming in the presence of pre-existing, but tolerated, DNA damage is aborted by the activation of a DNA damage response and p53-dependent apoptosis. -q Abrogation of p53 allows efficient reprogramming in the face of DNA damage and the generation of iPS cells carrying persistent DNA damage and chromosomal aberrations. -p53 downregulation improves reprogramming efficiency 20 . -FACS -western blotA subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\A subpopulation of adult skeletal muscle stem cells retains all template DNA strands after cell division.pdf" Abstract Satellite cells are adult skeletal muscle stem cells that are quiescent and constitute a poorly defined heterogeneous population. Using transgenic Tg:Pax7-nGFP mice, we show that Pax7-nGFPHi cells are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFPLo cells. Pax7-nGFPHi can give rise to Pax7-nGFPLo cells after serial transplantations. Proliferating Pax7-nGFPHi cells exhibit lower metabolic activity, and the majority performs asymmetric DNA segregation during cell division, wherein daughter cells retaining template DNA strands express stem cell markers. Using chromosome orientation-fluorescence in situ hybridization, we demonstrate that all chromatids segregate asymmetrically, whereas Pax7-nGFPLo cells perform random DNA segregation. Therefore, quiescent Pax7-nGFPHi cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFPHi cells generate distinct daughter cell fates by asymmetrically segregating template DNA strands to the stem cell. These findings provide major insights into the biology of stem cells that segregate DNA asymmetrically. Some notes -France Spain Switzerland paper -Shahragim Tajbakhsh is corresponding author -Cell paper -Some of these observations led to the ‘‘immortal DNA strands’’ hypoth- esis that postulated that stem cells retain old DNA strands of all chromosomes in the longer-lasting daughter stem cell, thereby avoiding a mutation load potentially arising from DNA replication. -Label Retention and Relative Pax7 Expression Identify a Subpopulation of Dormant Adult Muscle Stem Cells -Self-Renewal and Differentiation Potential of Serially Transplanted Subpopulations of Satellite Cells -Cells Performing TDSS Are Enriched in the Pax7 Hi Satellite Cell Subpopulation -Fate of Muscle Stem Cells Cosegregating Template DNA Strands -Template DNA Strand Cosegregation during Cell Division Engages All Chromatids -cell culture -FACS -satellite cell transplantation -gene expression analysis -immunohistochemistry -CO-FISHAging by telomere loss can be reversed "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Aging by telomere loss can be reversed.pdf" Abstract Recently in Nature, Jaskelioff et al. (2010) demonstrated that multiple aging phenotypes in a mouse model of accelerated telomere loss can be reversed within 4 weeks of reactivating telomerase. This raises the major question of whether physiological aging, likely caused by a combination of molecular defects, may also be reversible. Some notes -Cell stem cell preview -CNIO paper -she mentions Jaskelioff 2010 Nature paper: Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice --"C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Telomerase reactivation reverses tissue degeneration in aged telomerase-deficient mice.pdf" --Harvard Medical School papers -Tomas-Loba 2008 paper (cancer resistant mice with enhanced telomerase expression) --Telomerase Reverse Transcriptase Delays Aging in Cancer-Resistant Mice (CNIO paper) --"C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Telomerase Reversse Transcriptase Delays Aging in Cancer-Resistant Mice.pdf" --This is a big paper I missed because I was only looking at papers that were 2009 and after -q small molecule telomerase acti- vators have been reported recently and have demonstrated some preliminary health-span bene?cial effects in humansATR suppresses telomere fragility and recombination but is dispensable for elongation of short telomeres by telomerase "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-03-2014d1015\SIRT1 contributes to telomere maintenance and augments global homologous recombination.pdf" Telomere shortening caused by incomplete DNA replication is balanced by telomerase-mediated telomere extension, with evidence indicating that the shortest telomeres are preferred substrates in primary cells. Critically short telomeres are detected by the cellular DNA damage response (DDR) system. In budding yeast, the important DDR kinase Tel1 (homologue of ATM [ataxia telangiectasia mutated]) is vital for telomerase recruitment to short telomeres, but mammalian ATM is dispensable for this function. We asked whether closely related ATR (ATM and Rad3 related) kinase, which is important for preventing replicative stress and chromosomal breakage at common fragile sites, might instead fulfill this role. The newly created ATR-deficient Seckel mouse strain was used to examine the function of ATR in telomerase recruitment and telomere function. Telomeres were recently found to resemble fragile sites, and we show in this study that ATR has an important role in the suppression of telomere fragility and recombination. We also find that wild-type ATR levels are important to protect short telomeres from chromosomal fusions but do not appear essential for telomerase recruitment to short telomeres in primary mouse embryonic fibroblasts from the ATR-deficient Seckel mouse model. These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability. Some notes -These results reveal a previously unnoticed role for mammalian ATR in telomere protection and stability. -Generation of mice and cells doubly deficient in ATR and Terc -Severely decreased ATR levels in primary Seckel MEFs do not significantly impair elongation of short telomeres by telomerase -Severely decreased ATR levels augment telomere recombination in Seckel MEFs -Severely reduced ATR levels do not augment end-to-end fusions caused by short telomeres -ATR prevents telomeres from becoming fragile sites -Severe developmental defects in Seckel mice simultaneously abrogated for Terc -In conclusion, we show in this study that severe reduction in ATR levels does not impair telomerase recruitment to short telo- meres but increases telomere fragility and telomere recombina- tion, indicating that the important DDR checkpoint kinases ATM and ATR function differently in telomere maintenance in mam- mals and yeast. -western blot -trap assay -Q-FISH -Combined Q-FISH and SKY -Cytogenetic analyses -CO-FISH -Global SCE determinationBeyond average c potential for measurement of short telomeres Beyond average c potential for measurement of short telomeres "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Beyond average c potential for measurement of short telomeres.pdf" Abstract Abstract: The length of telomeres, and in particular the abundance of short teiomeres, has been proposed as a biomarlter of aging and of general health status. A wide variety of studies show the association of short telomeres with age related pathologies and cancer, as weli as with lifespan and mortality. These facts highlight the importance of measuring telomere length in human populations and by using reliable methods to uncover the association between telornere length and human disease. This reulew discusses the advantages and drawbacks of current teiornere length measurement methods. Most of these methods prouide mean telomere length ualues per cell or per sample and very few of them are able to measure the abundance of short telomeres, which are the ones indicatiue of telomere dysfunction. The information prouided by each method and their suitability for different studies is discussed here Some notes -impactaging journal -CNIO paper -show the association of short telomeres with age related pathologies and cancer, as well as with lifespan and mortality - very few of them are able to measure  the  abundance  of  short  telomeres,  which  are  the  ones  indicative  of  telomere  dysfunction -many different types of Q-FISH -telomere length measurement methods -It looks like "Improved HT Q-FISH" is the best -Fluorescence In Situ Hybridization (FISH) Methods -FISH uses fluorescent peptide nucleic acid (PNA) oligonucleotide probes -telomapping -Main telomere length measurement methods timeline -q Moreover, telomere length measured over time (longitudinal studies) may provide a valuable instrument to characterize positive or negative effects of several treatments or changes in lifestyle habits. - p16 and 53BP1 levels, to be able to define an “aging signature” -q Maria Blasco acts as an advisor and owns stock from LifeLength, SL, a biotech company based on telomere length determinations from biomedical purposes. BRCA2 acts as a RAD51 loader to facilitate telomere replication and capping "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-03-2014d1015\BRCA2 acts as a RAD51 loader to facilitate telomere replication and capping.pdf" The tumor suppressor protein BRCA2 is a key component of the homologous recombination pathway of DNA repair, acting as the loader of RAD51 recombinase at sites of double-strand breaks. Here we show that BRCA2 associates with telomeres during the S and G2 phases of the cell cycle and facilitates the loading of RAD51 onto telomeres. Conditional deletion of Brca2 and inhibition of Rad51 in mouse embryonic fibroblasts (MEFs), but not inactivation of Brca1, led to shortening of telomeres and accumulation of fragmented telomeric signals—a hallmark of telomere fragility that is associated with replication defects. These findings suggest that BRCA2-mediated homologous recombination reactions contribute to the maintenance of telomere length by facilitating telomere replication and imply that BRCA2 has an essential role in maintaining telomere integrity during unchallenged cell proliferation. Mouse mammary tumors that lacked Brca2 accumulated telomere dysfunction–induced foci. Human breast tumors in which BRCA2 was mutated had shorter telomeres than those in which BRCA1 was mutated, suggesting that the genomic instability in BRCA2-deficient tumors was due in part to telomere dysfunction. some notes -Conditional deletion of Brca2 and inhibition of Rad51 in mouse embryonic fibroblasts (MEFs), but not inactivation of Brca1, led to shortening of telomeres and accumulation of fragmented telomeric signals -Human breast tumors in which BRCA2 was mutated had shorter telomeres than those in which BRCA1 was mutated, suggesting that the genomic instability in BRCA2-deficient tumors was due in part to telomere dysfunction. -BRCA2 and RAD51 associate with telomeres during S phase -Telomere attrition due to BRCA2 deficiency in MEFs -Telomere length determines attrition induced by RAD51 loss -BRCA2 and RAD51 deletions induce telomere fragility -Damage response at uncapped telomeres in BRCA2-deficient cells -Telomere shortening in BRCA2-deficient human mammary tumors -Telomere homeostasis requires BRCA2 -BRCA2-mediated reactions in telomere replication and protection Chromatin regulation and non-coding RNAs at mammalian telomeres "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-01-2014d1112\Chromatin regulation and non-coding RNAs at mammalian telomeres.pdf" Abstract In eukaryotes, terminal chromosome repeats are bound by a specialized nucleoprotein complex that controls telomere length and protects chromosome ends from DNA repair and degradation. In mammals the “shelterin” complex mediates these central functions at telomeres. In the recent years it has become evident that also the heterochromatic structure of mammalian telomeres is implicated in telomere length regulation. Impaired telomeric chromatin compaction results in a loss of telomere length control. Progressive telomere shortening affects chromatin compaction at telomeric and subtelomeric repeats and activates alternative telomere maintenance mechanisms. Dynamics of chromatin structure of telomeres during early mammalian development and nuclear reprogramming further indicates a central role of telomeric heterochromatin in organismal development. In addition, the recent discovery that telomeres are transcribed, giving rise to UUAGGG-repeat containing TelRNAs/TERRA, opens a new level of chromatin regulation at telomeres. Understanding the links between the epigenetic status of telomeres, TERRA/TelRNA and telomere homeostasis will open new avenues for our understanding of organismal development, cancer and ageing. some notes -review paper -the recent discovery that telom- eres are transcribed, giving rise to UUAGGG-repeat containing TelRNAs/TERRA, opens a new level of chromatin regulation at telomeres. -Telomeres and shelterin -The signature of mammalian telomeric heterochromatin -DNA methylation at subtelomeric repeats -Epigenetic regulation of mammalian telomeres -Telomere repeat associated transcription (TERRA/TelRNAs) -Telomeric chromatin dynamics during differentiation and nuclear reprogramming -q indicates that telomere length and chromatin status is controlled by cell type speci?c programs -Outlook: implications of telomeric chromatin for human disease -Human population studies have recently linked environmental in?uences (smoking, obesity, or stress) with an accelerated rate of telomere shortening -Syndromes like aplastic anaemia (TERC, TERT), dyskeratosis congenita (DKC1, TERC) and idiopathic pulmonary ?brosis [132–134], but also mutations in various DNA repair genes [ataxia telangiectasia (ATM), Werner (WRN) and Bloom syndromes (BLM); Fanconi anaemia (Fanc genes) and Nijmegen breakage syn- drome (NBN)] are linked to an elevated risk to the development of premature ageing syndromes that such as premature loss of tissue renewal and bone marrow failure, leading to premature deathCohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Cohesin-SA1 deficiency drives aneuploidy and tumourigenesis in mice due to impaired replication of telomeres.pdf" Abstract Cohesin is a protein complex originally identi?ed for its role in sister chromatid cohesion, although increasing evidence portrays it also as a major organizer of interphase chromatin. Vertebrate cohesin consists of Smc1, Smc3, Rad21/Scc1 and either stromal antigen 1 (SA1) or SA2. To explore the functional speci?city of these two versions of cohesin and their relevance for embryonic development and cancer, we generated a mouse model de?cient for SA1. Complete ablation of SA1 results in embryonic lethality, while heterozygous animals have shorter lifespan and earlier onset of tumourigenesis. SA1-null mouse embryo- nic ?broblasts show decreased proliferation and increased aneuploidy as a result of chromosome segregation defects. These defects are not caused by impaired centromeric cohesion, which depends on cohesin-SA2. Instead, they arise from defective telomere replication, which requires cohesion mediated speci?cally by cohesin-SA1. We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis. Some notes -CNIO and New York (Albert Einstein College of Medicine) paper -EMBO journal -We propose a novel mechanism for aneuploidy generation that involves impaired telomere replication upon loss of cohesin-SA1, with clear implications in tumourigenesis. -Telomere-speci?c cohesion defects in SA1-null cells -Aberrant telomeres in cells lacking SA1 -De?cient telomere replication in SA1-null cells -Cohesin-SA1 is required for ef?cient telomere replication -FISH -knockout mice -chromosome spreads -FACS -quantitative RT-PCR -ChIP -RNAi -recombinant protein expression -live-cell imaging -histology and immunohistochemistry -carcinogen treatmentsConditional TRF1 knockout in the hematopoietic compartment leads to bone marrow failure and recapitulates clinical features of Dyskeratosis congenita "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Conditional TRF1 knockout in the hematopoietic compartment leads to bone marrow failure and recapitulates clinical features of Dyskeratosis congenita.pdf" Abstract TRF1 is part of the shelterin complex, which binds telomeres and it is essential for their protection. Ablation of TRF1 induces sister telomere fusions and aberrant numbers of telomeric signals associated with telomere fragility. Dyskeratosis congenita is characterized by a mucocutaneous triad, bone marrow failure (BMF), and presence of short telomeres because of mutations in telomerase. A subset of patients, however, show mutations in the shelterin component TIN2, a TRF1-interacting protein, presenting a more severe phenotype and presence of very short telomeres despite normal telomerase activity. Allelic variations in TRF1 have been found associated with BMF. To address a possible role for TRF1dysfunction in BMF, here we generated a mouse model with conditional TRF1deletion in the hematopoietic system. Chronic TRF1 deletion results in increased DNA damage and cellular senescence, but not increased apoptosis, in BM progenitor cells, leading to severe aplasia. Importantly, increased compensatory proliferation of BM stem cells is associated with rapid telomere shortening and further increase in senescent cells in vivo, providing a mechanism for the very short telomeres of human patients with mutations in the shelterin TIN2. Together, these results represent proof of principle that mutations in TRF1lead to the main clinical features of BMF. Some notes -CNIO paper -BLOOD journal -q Together, these results represent proof of principle that muta- tions in TRF1 lead to the main clinical features of BMF -q Dyskeratosis congenita (DKC) is considered to be paradigmatic of premature aging syndromes and it is characterized by the classic triad of bone marrow failure (BMF), skin abnormalities, and increased risk of cancer. -Bone marrow transplantation -G-CSF ELISA -FACS -colony-forming assay -Progressive TRF1 deletion signi?cantly reduces the number of HSC and progenitor cells and leads to increased compensatory proliferation -q Progressive TRF1 deletion leads to increased number of telomere-induced foci, increased p53-mediated induction of p21, and cellular senescence but no apoptosis --that's interesting that the cells don't undergo apoptosis -Long-term induction of TRF1 deletion leads to massive impaired overall survival because of BMF after 8 weeks and massive telomere shortening -Mice undergoing long-term TRF1 deletion undergo replicative senescence and exhaustion because of telomere shortening -q In summary, we provide the ?rst mouse model simulating DKC features caused by alteration of TRF1 and the conditional TRF1 deletion represents a useful model to further investigate the pathogenesis of DKCDifferent telomere-length dynamics at the inner cell mass versus established embryonic stem (ES) cells "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Different telomere-length dynamics at the inner cell mass versus established embryonic stem (ES) cells.pdf" Abstract Murine embryonic stem (ES) cells have unusually long telomeres, much longer than those in embryonic tissues. Here we address whether hyper-long telomeres are a natural property of pluripotent stem cells, such as those present at the blastocyst inner cell mass (ICM), or whether it is a characteristic acquired by the in vitro expansion of ES cells. We ?nd that ICM cells undergo telomere elongation during the in vitro derivation of ES-cell lines. In vivo analysis shows that the hyper-long telomeres of morula-injected ES cells remain hyper-long at the blastocyst stage and longer than telomeres of the blastocyst ICM. Telomere lengthening during derivation of ES-cell lines is concomitant with a decrease in heterochromatic marks at telomeres. We also found increased levels of the telomere repeat binding factor 1 (TRF1) telomere-capping protein in cultured ICM cells before telomere elongation occurs, coinciding with expression of pluripotency markers. These results suggest that high TRF1 levels are present in pluripotent cells, most likely to ensure pro?cient capping of the newly synthesized telomeres. These results highlight a previously unnoticed difference between ICM cells at the blastocyst and ES cells, and suggest that abnormally long telomeres in ES cells are likely to result from continuous telomere lengthening of proliferating ICM cells locked at an epigenetic state associated to pluripotency. Some notes -CNIO paper (PNAS) -q Murine embryonic stem (ES) cells have unusually long telomeres, much longer than those in embryonic tissues. -suggest that abnormally long telomeres in ES cells are likely to result from continuous telomere lengthening of proliferating ICM cells locked at an epigenetic state associated to pluripotency -q hyper-long telomeres in ES cell are aberrant and may result from the in vitro establishment and expansion of ES cells. -q Finally, the events described here associated to ES cell establishment, in- cluding the loss of heterochromatic marks, high levels of TRF1, and the elongation of telomeres, could also operate in the context of tumorigenesis to maintain cellular immortality.Genetic dissection of the mechanisms underlying telomere-associated diseases Impact of the TRF2 telomeric protein on mouse epidermal stem cells file located here "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-24-2014d0943\Genetic dissection of the mechanisms underlying telomere-associated diseases Impact of the TRF2 telomeric protein on mouse epidermal stem cells.pdf" notes -q Here, we show that K5TRF2 mice have a severe epidermal stem cell (ESC) dysfunction, which is reversed by abrogation of p53 in the absence of rescue of telomere length. -K15 a marker -q Remarkably, abrogation of p53 restored ESC functionality (mobilization and clonogenic activity) in K5TRF2 mice in the absence of telomere elongation. -label retaining (LRC) assay in K5TRF2 mice -in vitro clonogenic assays with newborn keratinocytes derived from wild-type K5TRF2 mice q indicating that transgenic TRF2 expression decreases the ex vivo proliferative capacity of ESCs. -Telomerase overexpression cannot rescue ESC defects in K5Tert/K5TRF2 mice -Altogether, these results indicate that persisting ESC defects in K5Tert/K5TRF2 mice are the consequence of a failure to rescue critically short telomeres by telomerase overexpression in these mice. -XPF deficiency rescues ESC proliferation defects in K5TRF2 mice -Q-FISH to measure telomere length -K5TRF2 mice have increased p53 expression and DNA damage in the skin -K5TRF2 mice have increased p21 and p19ARF expression in the skin (western blot) -p53 deletion rescues ESC defects in K5TRF2 skin keratinocytes in the absence of telomere elongation. -p53 deletion accelerates UV-induced skin cancer in K5TRF2 mice (makes sense since the cells would be dividing with short telomeres) -TRF2 is a central component of the shelterin complex that binds to telomeres, and has essential roles in the protection of chromosome ends and regulation of telomere length -confocal microscopyGenetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\Genetic inactivation of Cdk7 leads to cell cycle arrest and induces premature aging due to adult stem cell exhaustion.pdf" Abstract Cyclin-dependent kinase (Cdk)7, the catalytic subunit of the Cdk-activating kinase (CAK) complex has been implicated in the control of cell cycle progression and of RNA polymerase II (RNA pol II)-mediated transcription. Genetic inactivation of the Cdk7 locus revealed that whereas Cdk7 is completely dispensable for global transcription, is essential for the cell cycle via phosphorylation of Cdk1 and Cdk2. In vivo, Cdk7 is also indispensable for cell proliferation except during the initial stages of embryonic development. Interestingly, widespread elimination of Cdk7 in adult tissues with low proliferative indexes had no phenotypic consequences. However, ablation of conditional Cdk7 alleles in tissues with elevated cellular turnover led to the efficient repopulation of these tissues with Cdk7-expressing cells most likely derived from adult stem cells that may have escaped the inactivation of their targeted Cdk7 alleles. This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age-related phenotypes, including telomere shortening and early death. Some Notes -CNIO EMBO Journal -corresponding author: D Santamaria or M Barbacid - This process, a physiological attempt to maintain tissue homeostasis, led to the attrition of adult stem cell pools and to the appearance of age-related phenotypes, including telomere shortening and early death -Cdk7 is required for cell proliferation but not for global transcription -Cdk7 is essential for activation of cell cycle Cdks -Cdk7 is required for T-loop phosphorylation of cell cycle Cdks -Restoration of E2F-dependent transcription rescues proliferation of Cdk7-defective cells -Cdk7 de?ciency causes early embryonic lethality -Cdk7 is required for skin development -Deletion of Cdk7 in adult mice -Loss of Cdk7 expression in actively dividing tissues results in accelerated aging -Stem cell exhaustion in Cdk7 depleted skin -q The maintenance of normal physiological parameters by critical tissues such as liver, kidney or the endocrine pancreas in the absence of Cdk7 suggests that Cdk7 inhibitors may not cause a signi?cant toxicity, providing that they are not administered for long periods of time. Yet, the potential use of such inhibitors may depend on the relative ability of adult stem cells to replenish highly proliferating tissues versus cancer stem cells to replace Cdk7-de?cient tumour cells. -genotyping -RT PCR -DEXA -immunofluorescence -cell culture -Gene expression analysis DNA microarray -Histopathology -western blotBp1Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Genome-wide CTCF distribution in vertebrates defines equivalent sites that aid the identification of disease-associated genes.pdf" Abstract Many genomic alterations associated with human diseases localize in noncoding regulatory elements located far from the promoters they regulate, making it challenging to link noncoding mutations or risk-associated variants with target genes. The range of action of a given set of enhancers is thought to be defined by insulator elements bound by the 11 zinc-finger nuclear factor CCCTC-binding protein (CTCF). Here we analyzed the genomic distribution of CTCF in various human, mouse and chicken cell types, demonstrating the existence of evolutionarily conserved CTCF-bound sites beyond mammals. These sites preferentially flank transcription factor–encoding genes, often associated with human diseases, and function as enhancer blockers in vivo, suggesting that they act as evolutionarily invariant gene boundaries. We then applied this concept to predict and functionally demonstrate that the polymorphic variants associated with multiple sclerosis located within the EVI5 gene impinge on the adjacent gene GFI1. Some notes -nature structural & molecular biology paper -Spain, Mexico, NIH, Portugal paper -casfer and gomska are corresponding others -11 zinc-finger nuclear factor CCCTC-binding protein (CTCF) -CTCF occupies syntenic positions in vertebrate genomes -Genomic features of CONSYN sites - CONSYN-CTCF sites preferentially flank transcription factors involved in developmental processes -CONSYN sites predict GFI1 association to multiple sclerosis - increased risk of multiple sclerosis located in the GFI1–EVI5 genomic region -ChIP -sequencing -motif analysis -gene ontology analysis -enhancer-blocking assay -repressor luciferase assays -morpholino injections -chromosome conformation capture assayIdentification of novel pathways involved in the pathogenesis of human adamantinomatous craniopharyngioma "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Identification of novel pathways involved in the pathogenesis of human adamantinomatous craniopharyngioma.pdf" Abstract Activating mutations in the gene encoding ß-catenin have been identified in the paediatric form of human craniopharyngioma (adamantinomatous craniopharyngioma, ACP), a histologically benign but aggressive pituitary tumour accounting for up to 10% of paediatric intracranial tumours. Recently, we generated an ACP mouse model and revealed that, as in human ACP, nucleocytoplasmic accumulation of ß-catenin (ß-catnc) and over-activation of the Wnt/ß-catenin pathway occurs only in a very small proportion of cells, which form clusters. Here, combining mouse genetics, fluorescence labelling and flow-sorting techniques, we have isolated these cells from tumorigenic mouse pituitaries and shown that the ß-catnc cells are enriched for colony-forming cells when cultured in stem cell-promoting media, and have longer telomeres, indicating shared properties with normal pituitary progenitors/stem cells (PSCs). Global gene profiling analysis has revealed that these ß-catnc cells express high levels of secreted mitogenic signals, such as members of the SHH, BMP and FGF family, in addition to several chemokines and their receptors, suggesting an important autocrine/paracrine role of these cells in the pathogenesis of ACP and a reciprocal communication with their environment. Finally, we highlight the clinical relevance of these findings by showing that these pathways are also up-regulated in the ß-catnc cell clusters identified in human ACP. As well as providing further support to the concept that pituitary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours. Some notes -Acta Neuropatholology Journal -London Spain North Carolina paper -q As well as providing further support to the concept that pitu- itary stem cells may play an important role in the oncogenesis of human ACP, our data reveal novel disease biomarkers and potential pharmacological targets for the treatment of these devastating childhood tumours -Martinez-Barbera in London was corresponding author -Immuno-FISH -FACS -Cell culture Impact papers on aging in 2009 "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-31-2014d1002\Impact papers on aging in 2009.pdf" Abstract The editorial board of Aging reviews research papers published in 2009, which they believe have or will have a significant impact on aging research. Among many others, the topics include genes that accelerate aging or in contrast promote longevity in model organisms, DNA damage responses and telomeres, molecular mechanisms of life span extension by calorie restriction and pharmacologic interventions into aging. The emerging message in 2009 is that aging is not random but determined by a genetically-regulated longevity network and can be decelerated both genetically and pharmacologically. some notes DNA damage response and aging Mitochondria, oxidative stress and aging Calorie restriction (CR) Pharmacologic intervention Stem cells and aging Nuclear reprogramming and senescence Genetics of aging Autophagy Post-transcriptional gene regulation and aging Circadian clock Cancer and agingIncreased telomere fragility and fusions resulting from TRF1 deficiency lead to degenerative pathologies and increased cancer in mice "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-30-2014d1027\Increased telomere fragility and fusions resulting from TRF1 deficiency lead to degenerative pathologies and increased cancer in mice.pdf" Abstract The telomere repeat-binding factor 1 (TERF1, referred to hereafter as TRF1) is a component of mammalian telomeres whose role in telomere biology and disease has remained elusive. Here, we report on cells and mice conditionally deleted for TRF1. TRF1-deleted mouse embryonic fibroblasts (MEFs) show rapid induction of senescence, which is concomitant with abundant telomeric g-H2AX foci and activation of the ATM/ATR downstream checkpoint kinases CHK1 and CHK2. DNA damage foci are rescued by both ATM and ATM/ATR inhibitors, further indicating that both signaling pathways are activated upon TRF1 deletion. Abrogation of the p53 and RB pathways bypasses senescence but leads to chromosomal instability including sister chromatid fusions, chromosome concatenation, and occurrence of multitelomeric signals (MTS). MTS are also elevated in ATR-deficient MEFs or upon treatment with aphidicolin, two conditions known to induce breakage at fragile sites, suggesting that TRF1-depleted telomeres are prone to breakage. To address the impact of these molecular defects in the organism, we deleted TRF1 in stratified epithelia of TRF1 D/D K5-Cre mice. These mice die perinatally and show skin hyperpigmentation and epithelial dysplasia, which are associated with induction of telomere-instigated DNA damage, activation of the p53/p21 and p16 pathways, and cell cycle arrest in vivo. p53 deficiency rescues mouse survival but leads to development of squamous cell carcinomas, demonstrating that TRF1 suppresses tumorigenesis. Together, these results demonstrate that dysfunction of a telomere-binding protein is sufficient to produce severe telomeric damage in the absence of telomere shortening, resulting in premature tissue degeneration and development of neoplastic lesions. Some notes -constructed target vectors -cell fractionation and immunoblotting -IF-FISH -TRF1 knockout mice -retroviral infections -FACS -Q-FISH -Western blotsIsolation and in vitro expansion of human colonic stem cells "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Isolation and in vitro expansion of human colonic stem cells.pdf" Abstract Here we describe the isolation of stem cells of the human colonic epithelium. Differential cell surface abundance of ephrin type-B receptor 2 (EPHB2) allows the purification of different cell types from human colon mucosa biopsies. The highest EPHB2 surface levels correspond to epithelial colonic cells with the longest telomeres and elevated expression of intestinal stem cell (ISC) marker genes. Moreover, using culturing conditions that recreate the ISC niche, a substantial proportion of EPHB2-high cells can be expanded in vitro as an undifferentiated and multipotent population. Some notes -nature medicine brief communication paper -Spain Netherland Japan paper -corresponding author: aduard batlle -Collectively, these data represent what is to our knowledge the first isolation, characterization and in vitro expansion of a cell popula- tion largely enriched in human CoSCs. -CoSc: colon stem cells Localization-dependent and -independent roles of SLX4 in regulating telomeres "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\Localization-dependent and -independent roles of SLX4 in regulating telomeres.pdf" Abstract SLX4, a scaffold for structure-specific DNA repair nucleases, is important for several types of DNA repair. Many repair proteins bind to sites of DNA damage, resulting in subnuclear "foci," but SLX4 forms foci in human cells even without DNA damage. Using several approaches, we show that most, but not all, SLX4 foci localize to telomeres in a range of human cell lines irrespective of the mechanisms used to maintain telomere length. The SLX1 Holliday-junction-processing enzyme is recruited to telomeres by SLX4, and SLX4, in turn, is recruited by a motif that binds to the shelterin subunit TRF2 directly. We also show that TRF2-dependent recruitment of SLX4 prevents telomere damage. Furthermore, SLX4 prevents telomere lengthening and fragility in a manner that appears to be independent of telomere association. These findings reveal that SLX4 plays multiple roles in regulating telomere homeostasis. Some notes -Cell reports journal -Scotland and CNIO -correspondence with John Rouse -The Endogenous SLX4 Complex Localizes at Telomeres -SLX4 Binds to Telomeres in Cells Differing in Mechanisms of Telomere Length Maintenance -SLX4 Has a TRF2-Binding Motif -The SLX4 Complex Is Recruited to Telomeres by TRF2 in Human Cells -SLX4 Localizes at and Repairs DNA Damage -Localization-Dependent and -Independent Roles for SLX4 in Promoting Telomere Homeostasis - q From this perspective, SLX4 might mediate ‘‘telo- mere trimming,’’ a mechanism that has been proposed to pre- vent overlengthening of telomeres -q The consequences of overlong telomeres for cell function and organism function are not clear, but this will be interesting to study. -huh I hadn't heard that telomeres could be too long before Independently of TRF2 -immunofluorescence -immunoprecipitationMammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-03-2014d1015\Mammalian Rap1 controls telomere function and gene expression through binding to telomeric and extratelomeric sites.pdf" Abstract Rap1 is a component of the shelterin complex at mammalian telomeres, but its in vivo role in telomere biology has remained largely unknown to date. Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. We generated cells and mice deleted for Rap1; mice with Rap1 deletion in stratified epithelia were viable but had shorter telomeres and developed skin hyperpigmentation in adulthood. By performing chromatin immunoprecipitation coupled with ultrahigh-throughput sequencing, we found that Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG)2 consensus motif. Extratelomeric Rap1-binding sites were enriched at subtelomeric regions, in agreement with preferential deregulation of subtelomeric genes in Rap1-deficient cells. More than 70% of extratelomeric Rap1-binding sites were in the vicinity of genes, and 31% of the genes deregulated in Rap1-null cells contained Rap1-binding sites, suggesting a role for Rap1 in transcriptional control. These findings place a telomere protein at the interface between telomere function and transcriptional regulation. Some notes -Here we show that Rap1 deficiency is dispensable for telomere capping but leads to increased telomere recombination and fragility. -Rap1 binds to both telomeres and to extratelomeric sites through the (TTAGGG) 2 consensus motif -suggesting a role for Rap1 in transcriptional control. -Rap1 abrogation does not disrupt binding of other shelterins to telomeres -No fusions but increased telomere fragility and recombination in Rap1-null cells -Mice with Rap1 deletion in stratified epithelia are viable but develop skin hyperpigmentation in adulthood -Preferential derepression of subtelomeric genes in Rap1 ?/? cells -Rap1 transcriptional networks include cell adhesion, cancer and metabolism -Rap1 ?/? K5-Cre females show increased body weight -Mapping in vivo Rap1-binding sites by using ChIP sequencing -Identification of a consensus Rap1-binding sequence to extratelomeric sites -me: it's interesting that Rap1 binds to more than just telomere sequences -our results provide evi- dence for a shelterin component (rap1) influencing gene transcription through binding to non-telomeric sites -Future studies merit the identification of factors responsible for Rap1 recruit- ment to non-TTAGGG-rich chromatin regions. -constructing a vector -knockout mice -immunoprecipitation -proliferation assay -flow cytometry -RT-PCR -Q-FISH -ChIP assay -B1-SINE Cobra analysis for global DNA methylation -Gene expression analysis -ChIP -qPCR -Luciferase assayMSH2 deficiency abolishes the anticancer and pro-aging activity of short telomeres "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-30-2014d1027\MSH2 deficiency abolishes the anticancer and pro-aging activity of short telomeres.pdf" { Mutations in the mismatch repair (MMR) pathway occur in human colorectal cancers with microsatellite instability. Mounting evidence suggests that cell-cycle arrest in response to a number of cellular stresses, including telomere shortening, is a potent anticancer barrier. The telomerase-deficient mouse model illustrates the anticancer effect of cell-cycle arrest provoked by short telomeres. Here, we describe a role for the MMR protein, MSH2, in signaling cell-cycle arrest in a p21/p53-dependent manner in response to short telomeres in the context of telomerase- deficient mice. In particular, progressively shorter telomeres at successive generations of MSH2 –/– Terc –/–- mice did not suppress cancer in these mice, indicating that MSH2 deficiency abolishes the tumor suppressor activity of short telomeres. Interestingly, MSH2 deficiency prevented degenerative pathologies in the gastrointestinal tract of MSH2 –/– Terc –/– mice concomitant with a rescue of pro- liferative defects. The abolishment of the anticancer and pro-aging effects of short telomeres provoked by MSH2 abrogation was independent of changes in telomere length. These results highlight a role for MSH2 in the organismal response to dysfunctional telomeres, which in turn may be important in the pathobiology of human cancers bearing mutations in the MMR pathway. Key words: aging; cancer; mouse models; MMR; telomeres. } some highlights and notes -The lifespan of mice lacking MSH2 does not decrease with successive generations of Terc deficiency -MSH2 abrogation rescues aging pathologies associated to short telomeres ^this seems surprising to me -MSH2 abrogation largely abolishes the tumor suppressor activity of short telomeres ^so there were more tumors -MSH2 abrogation rescues cell-cycle arrest but not apoptosis provoked by short telomeres -MSH2 deficiency does not prevent telomere shortening in Terc –/– mice -Ki67 positive cells (Ki67 necessary for cellular proliferation) -MSH2 deficiency does not rescue telomere-associated chromosomal instability in Terc-deficient mice -MSH2 deficiency does not lead to increased telomere recombination -MSH2 deficiency results in an attenuated p53 and p21 induction in response to short telomeres -MMR: mismatch repair -telomere length analysis by Q-FISH -TRF -histopathology and immunohistochemistry -Western blotp53 isoforms regulate aging- and tumor-associated replicative senescence in T lymphocytes "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-08-2014d0943\p53 isoforms regulate aging- and tumor-associated replicative senescence in T lymphocytes.pdf" Abstract Cellular senescence contributes to aging and decline in tissue function. p53 isoform switching regulates replicative senescence in cultured fibroblasts and is associated with tumor progression. Here, we found that the endogenous p53 isoforms ?133p53 and p53ß are physiological regulators of proliferation and senescence in human T lymphocytes in vivo. Peripheral blood CD8+ T lymphocytes collected from healthy donors displayed an age-dependent accumulation of senescent cells (CD28-CD57+) with decreased ?133p53 and increased p53ß expression. Human lung tumor-associated CD8+ T lymphocytes also harbored senescent cells. Cultured CD8+ blood T lymphocytes underwent replicative senescence that was associated with loss of CD28 and ?133p53 protein. In poorly proliferative, ?133p53-low CD8+CD28- cells, reconstituted expression of either ?133p53 or CD28 upregulated endogenous expression of each other, which restored cell proliferation, extended replicative lifespan and rescued senescence phenotypes. Conversely, ?133p53 knockdown or p53ß overexpression in CD8+CD28+ cells inhibited cell proliferation and induced senescence. This study establishes a role for ?133p53 and p53ß in regulation of cellular proliferation and senescence in vivo. Furthermore, ?133p53-induced restoration of cellular replicative potential may lead to a new therapeutic paradigm for treating immunosenescence disorders, including those associated with aging, cancer, autoimmune diseases, and HIV infection. Some notes -NIH CNIO, Czech republic, Singapore -Journal of clinical investigation -corresponding author is Curtis Harris at NIH -Furthermore, ?133p53-induced restora- tion of cellular replicative potential may lead to a new therapeutic paradigm for treating immunosenescence disorders, including those associated with aging, cancer, autoimmune diseases, and HIV infection. -In vivo accumulation of senescent CD8 + T lymphocytes during physiological aging -Reduced ?133p53 and elevated p53ß expression in senescent CD8 + blood T lymphocytes in vivo -Senescence-associated phenotypes and p53 isoform expression profile in tumor-associated CD8 + T lymphocytes -In vitro replicative senescence of CD8 + T lymphocytes reproduces CD28 loss and diminished ?133p53 expression observed in vivo -Reconstitution of either CD28 or ?133p53 restores cell proliferation, extends replicative lifespan, and rescues senescence phenotypes in CD8 + CD28 – T lymphocytes -Knockdown of ?133p53 or overexpression of p53ß induces cellular senes- cence in CD8 + CD28 + T lymphocytes. -q Since ?133p53 is a natural isoform of p53 and is physiologically expressed in normal prolifer- ative cells at high levels, enhanced expression of ?133p53 may lead to a safe method for functional restoration of CD8 + T lymphocytes with minimum concern for malignant transformation -cell culture -FACS -proliferation assay -immunoblot -immunohistochemistry -HT Q-FISH -lentiviral transduction -SA-beta-gal assay -siRNA knockdown -RT-PCRp53 Prevents entry into mitosis with uncapped telomeres "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-03-2014d1015\p53 Prevents entry into mitosis with uncapped telomeres.pdf" Abstract Telomeres are protected by capping structures consisting of core protein complexes that bind with sequence specificity to telomeric DNA (reviewed in [1]). In their absence, telomeres trigger a DNA damage response, materialized in accumulation at the telomere of damage response proteins, e.g., phosphorylated histone H2AX (?H2AX), into telomere-dysfunction-induced foci [2 and 3]. Telomere uncapping occurs transiently in every cell cycle in G2 [4], following DNA replication, but little is known about how protective structures are reassembled or whether this process is controlled by the cell-cycle surveillance machinery. Here, we report that telomere capping is monitored at the G2/M transition by the p53/p21 damage response pathway. Unlike their wild-type counterparts, human and mouse cells lacking p53 or p21 progress into mitosis prematurely with persisting uncapped telomeres. Furthermore, artificially uncapped telomeres delay mitotic entry in a p53- and p21-dependent manner. Uncapped telomeres that persist in mitotic p53-deficient cells are shorter than average and religate to generate end-to-end fusions. These results suggest that a p53-dependent pathway monitors telomere capping after DNA replication and delays G2/M progression in the presence of unprotected telomeres. This mechanism maintains a cell-cycle stage conducive for capping reactions and prevents progression into stages during which uncapped telomeres are prone to deleterious end fusions. -Mitotic TIFs Mark Short, Uncapped Telomeres -The p53/p21 Pathway Prevents Mitotic Entry with Uncapped Telomeres and Telomere Fusion -Uncapped Telomeres Trigger ATM- and p53/p21-Dependent G2/M Arrest -immunofluorescence -FISH -Q-FISHPersonal omics profiling reveals dynamic molecular and medical phenotypes "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\Personal omics profiling reveals dynamic molecular and medical phenotypes.pdf" Abstract Personalized medicine is expected to benefit from combining genomic information with regular monitoring of physiological states by multiple high-throughput methods. Here, we present an integrative personal omics profile (iPOP), an analysis that combines genomic, transcriptomic, proteomic, metabolomic, and autoantibody profiles from a single individual over a 14 month period. Our iPOP analysis revealed various medical risks, including type 2 diabetes. It also uncovered extensive, dynamic changes in diverse molecular components and biological pathways across healthy and diseased conditions. Extremely high-coverage genomic and transcriptomic data, which provide the basis of our iPOP, revealed extensive heteroallelic changes during healthy and diseased states and an unexpected RNA editing mechanism. This study demonstrates that longitudinal iPOP can be used to interpret healthy and diseased states by connecting genomic information with additional dynamic omics activity. Some notes -Stanford Yale CNIO -Cell paper -lots of authors on this paper -dynamic omics activity. -q Personalized medicine is expected to bene?t from combining genomic information with regular moni- toring of physiological states by multiple high- throughput methods -overview of personal omics profiling -this is pretty neat paper and a glimpse into the future. It looks at all types of omic data for one individual over 14 months -Whole-Genome Sequencing -WGS-Based Disease Risk Evaluation -Medical Phenotypes Monitoring -Dynamic Omics Analysis: Integrative Omics Pro?ling of Molecular Responses -Dynamic Omics Analysis: Extensive Heteroallelic Variation and RNA Editing -miRNA Variant Analysis -To our knowledge, our study is the ?rst to perform extensive personal iPOP of an individual through healthy and diseased states. It revealed extensive complex and dynamic changes in the omics pro?les, especially in the transcriptomes, between healthy states and viral infections, and between nondiabetic and diabetic states. -WGS . . .sequencing -cytokine assayPotential of telomerase activation in extending health span and longevity "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\Potential of telomerase activation in extending health span and longevity.pdf" Abstract The progressive increase in the elderly population worldwide has resulted in higher numbers of individuals affected by age-associated diseases, such as neurodegenerative and heart diseases, metabolic impairment, or cancer, with the subsequent burden for national health systems. Therapeutic interventions aimed to increase the quality of life at advanced age are visualized as important demands for the future, both at the level of individuals and society. Novel advances in telomerase function from several independent laboratories have resulted in potential new therapeutic strategies which appear as promising new venues to prevent cellular and tissue dysfunction and organismal decline, thereby increasing the so-called “health span”. Here, we analyze these recent advances. Some notes -Bernardes de Jesus and Maria Blasco paper (CNIO paper) -Current opinion in Cell biology paper -pathways involved in genome stability, nutrient sensing, oxidative damage balance, and growth, seem to be central in aging modu- lation -this looks like a really nice review and opinion paper -Telomerase, DNA damage, and aging -Telomerase phenotypes -Anti-aging role of telomerase -Anti-aging role of telomerase -Perspectives for a healthy life -Several independent works demonstrated that telomerase expression through genetic modi?cations, viral delivery or chemical acti- vation result in a signi?cant rescue of age-related path- ologiesRAP1 protects from obesity through its extratelomeric role regulating gene expression "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\RAP1 protects from obesity through its extratelomeric role regulating gene expression.pdf" Abstract RAP1 is part of shelterin, the protective complex at telomeres. RAP1 also binds along chromosome arms, where it is proposed to regulate gene expression. To investigate the nontelomeric roles of RAP1 in vivo, we generated a RAP1 whole-body knockout mouse. These mice show early onset of obesity, which is more severe in females than in males. Rap1-deficient mice show accumulation of abdominal fat, hepatic steatosis, and high-fasting plasma levels of insulin, glucose, cholesterol, and alanine aminotransferase. Gene expression analyses of liver and visceral white fat from Rap1-deficient mice before the onset of obesity show deregulation of metabolic programs, including fatty acid, glucose metabolism, and PPARa signaling. We identify Ppara and Pgc1a as key factors affected by Rap1 deletion in the liver. We show that RAP1 binds to Ppara and Pgc1a loci and modulates their transcription. These findings reveal a role for a telomere-binding protein in the regulation of metabolism. Some notes -CNIO and NIH paper -Cell journal - we generated a RAP1 whole-body knockout mouse. -These mice show early onset of obesity, which is more severe in females than in males -These ?ndings reveal a role for a telomere-binding protein in the regulation of metabolism. -Generation of Whole-Body Rap1 -De?cient Mice -Rap1 -De?cient Mice Are Viable but Show an Early Onset of Obesity -Rap1 -De?cient Mice Accumulate More Fat in Visceral Tissues and Show Signs of Liver Steatosis and In?ammation -Rap1 -De?cient Mice Are Glucose Resistant and Show Some Signs of Metabolic Syndrome -High-Fat Diet Further Aggravates Obesity and Diabetes in Rap1 -De?cient Females -Metabolic Alterations in Rap1 -De?cient Tissues Occur in the Absence of Changes in Telomere Length and in the Absence of Telomere Damage - Enhanced Weight Gain and Glucose Resistance in Rap1 Null Females Subjected to a HFD -RAP1 De?ciency Affects Metabolic Transcriptional Networks before the Onset of Obesity -Decreased mRNA and Protein Expression of Ppara and Pgc1a in Rap1 -De?cient Livers -Defective Expression of PPAR a Target Genes in Rap1 - De?cient Livers upon Fasting -RAP1 Binds to Ppara and Pgc1a Loci -RAP1 Regulates Ppara and Pgc1a Transcription -In conclusion, we demonstrate here that RAP1 serves as a transcriptional regulator that controls the capacity of down- stream metabolic pathways critical for metabolic maturation -knockout mice -ELISA -gene expression analysis -Luciferase and ChIP AssaysRole of shelterin in cancer and aging "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-01-2014d1112\Role of shelterin in cancer and aging.pdf" Abstract Mammalian telomeres are formed by tandem repeats of the TTAGGG sequence bound by a specialized six-protein complex known as shelterin, which has fundamental roles in the regulation of telomere length and telomere capping. In the past, the study of mice genetically modified for telomerase components has been instrumental to demonstrate the role of telomere length in cancer and aging. Recent studies using genetically modified mice for shelterin proteins have highlighted an equally important role of telomere-bound proteins in cancer and aging, even in the presence of proficient telomerase activity and normal telomere length. In this review, we will focus on recent findings, suggesting a role of shelterin components in cancer and aging. some notes -review -Telomeric DNA and its shield -Lessons learned from telomerase-de?cient and telomerase over-expressing mice -nice table of different mouse models and knockouts to study telomerase and the shelterin components -Mouse models to understand the role of shelterin proteins in cancer and aging -TRF1 -some cases of premature aging in human syndromes have been linked to shelterin mutations -the length of telomeres mat- ters as long as some of the shelterin components are bound to them Short telomeres are frequent in hereditary breast tumors and are associated with high tumor grade "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\Short telomeres are frequent in hereditary breast tumors and are associated with high tumor grade.pdf" Abstract Telomere shortening is a common event involved in malignant transformation. Critically short telomeres may trigger chromosomal aberrations and produce genomic instability leading to cancer development. Therefore, telomere shortening is a frequent molecular alteration in early stages of many epithelial tumors and in breast cancer correlates with stage and prognosis. A better understanding of the involvement of short telomeres in tumors may have a significant impact on patient management and the design of more specific treatments. To understand the role of telomere length (TL) in breast cancer etiology we measured the length of individual telomere signals in single cells by using quantitative telomere in situ hybridization in paraffin-embedded tissue from hereditary and sporadic breast cancers. A total of 104 tumor tissue samples from 75 familial breast tumors (BRCA1, n = 14; BRCA2, n = 13; non-BRCA1/2, n = 48) and 29 sporadic tumors were analyzed. Assessment of telomere signal intensity allowed estimation of the mean TL and related variables, such as percentage of critically short telomeres and percentage of cells with short telomeres. These data were correlated with the immunohistochemical expression of molecular breast cancer markers. Hereditary BRCA1, BRCA2, and non-BRCA1/2 tumors were characterized by shorter TL comparing to sporadic tumors. Considering all tumors, tumor grade was a strong risk factor determining the proportion of short telomeres or short telomere cells. Moreover, some histopathological features appeared to be differentially associated to hereditary or sporadic subgroups. Short telomeres correlated with ER-negative tumors in sporadic cases but not in familial cases, whereas a high level of apoptosis was associated with shorter telomeres in hereditary BRCA1 and BRCA2 tumors. In addition, TL helped to define a subset of non-BRCA1/2 tumors with short telomeres associated with increased expression of antiapoptotic proteins. These findings highlight the potential interest of TL measurements as markers of aggressiveness in breast cancer. Some notes -breast cancer research treatment -CNIO -B Martinez-Delgado is corresponding author -Q-FISH -immunohistochemistry -Telomere length distribution in hereditary and sporadic breast tumors -shorter telomeres in hereditary breast cancer, -Histopathological variables and telomere length measurements -Histopathological variables speci?cally associated with shorter telomeres in hereditary or sporadic tumors - We found that hereditary breast tumor samples showed shorter TL compared to sporadic breast cancer samples. -Q-FISH is a useful technique that in combi- nation with immunohistochemistry may differentiate spe- ci?c subsets of tumors with short telomeres -q In conclusion, telomere dysfunction is frequently found in hereditary tumors and may be a marker of tumor aggressiveness in breast cancer patients Shorter telomere length is associated with increased ovarian cancer risk in both familial and sporadic cases asu doesn't have access to this articleSIRT1 contributes to telomere maintenance and augments global homologous recombination "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-03-2014d1015\SIRT1 contributes to telomere maintenance and augments global homologous recombination.pdf" Abstract Yeast Sir2 deacetylase is a component of the silent information regulator (SIR) complex encompassing Sir2/Sir3/Sir4. Sir2 is recruited to telomeres through Rap1, and this complex spreads into subtelomeric DNA via histone deacetylation. However, potential functions at telomeres for SIRT1, the mammalian orthologue of yeast Sir2, are less clear. We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1super) mouse models. Our results indicate that SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging, an effect dependent on telomerase activity. Using chromatin immunoprecipitation assays, we find that SIRT1 interacts with telomeric repeats in vivo. In addition, SIRT1 overexpression increases homologous recombination throughout the entire genome, including telomeres, centromeres, and chromosome arms. These findings link SIRT1 to telomere biology and global DNA repair and provide new mechanistic explanations for the known functions of SIRT1 in protection from DNA damage and some age-associated pathologies. some notes -We studied both loss of function (SIRT1 deficient) and gain of function (SIRT1 super ) mouse models - SIRT1 is a positive regulator of telomere length in vivo and attenuates telomere shortening associated with aging -SIRT1 overexpression increases homologous recombination throughout the entire genome -SIRT1 levels influence telomere length in mouse embryonic fibroblasts (MEFs) -Increased SIRT1 expression attenuates telomere erosion with age in adult tissues -SIRT1 effects on telomere length are largely dependent on telomerase activity -Increased SIRT1 expression augments homologous recombination at telomeres, centromeres, and chromosome arms -SIRT1 deficiency triggers a DNA damage response at chromosome ends -SIRT1 prevents telomere fragility -SIRT1-mediated deacetylation of telomeric and pericentromeric regions -SIRT1 specifically binds to telomeric repeats in vivo in induced pluripotent stem (iPS) cells -However, the molecular mechanisms by which SIRT1 impacts on CR, lifespan, and health span are still largely un- known -In summary, the findings described in this study demon - strate that increased expression of SIRT1, the closest mam- malian orthologue of yeast Sir2, improves telomere length maintenance in vivo and significantly increases recombination frequencies at telomeres, centromeres, and chromosome arms -cell culture -TRAP assay -immunoblotting -immunofluorescence -Q-FISH -TRF analysis -B1-SINE Cobra analysis for global DNA methylation -ChIP assay -CO-FISHSuv4-20h Abrogation Enhances Telomere Elongation during Reprogramming and Confers a Higher Tumorigenic Potential to iPS Cells Telomerase at the intersection of cancer and aging "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-08-2014d0943\Telomerase at the intersection of cancer and aging.pdf" "C:\Users\kurtw_000\Box Sync\DocDR\2013\11-22-13\Telomerase at the intersection of cancer and aging.pdf" "C:\Users\kurtw_000\Box Sync\DocDR\2013\11-22-13\Telomerase at the intersection of cancer and aging (annotated).pdf" Abstract Although cancer and aging have been studied as independent diseases, mounting evidence suggests that cancer is an aging-associated disease and that cancer and aging share many molecular pathways. In particular, recent studies validated telomerase activation as a potential therapeutic target for age-related diseases; in addition, abnormal telomerase expression and telomerase mutations have been associated with many different types of human tumor. Here, we revisit the connection between telomerase and cancer and aging in light of recent findings supporting a role for telomerase not only in telomere elongation, but also in metabolic fitness and Wnt activation. Understanding the physiological impact of telomerase regulation is fundamental given the therapeutic strategies that are being developed that involve telomerase modulation. Some notes -Bernardes de Jesus and Maria Blasco paper -CNIO paper -Cell review paper -suggests that can- cer is an aging-associated disease and that cancer and aging share many molecular pathways. -Telomerase defects may lead to aging and cancer -Telomerase as a key factor that regulates aging -Telomerase regulation in cancer -Telomerase in stem cells -Therapies based on telomerase: therapeutic value and future perspectives -q It seems hazardous to use the lack of tumorigenesis in mice as evidence for the safety of pro- telomerase therapies in humans, because telomerase is differentially regulated in these organisms -As a starting point for translating these ?ndings to the clinic, telomerase activation is likely to be tested ?rst for treatment of telomere syndromes -q Indeed, a change of paradigm seems to be occurring in telomerase biology, with a switch from viewing telomerase as fueling cancer to reversing aging. -q Supporting this notion are novel telome- rase activators [120,127,128], some of which are commer- cially available and used as anti-aging supplements Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer "C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2013\9-15-13\Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer.pdf" "C:\Users\kurtw_000\Documents\kurt\storage\CIM Research Folder\DR\2013\9-15-13\Telomerase gene therapy in adult and old mice delays aging and increases longevity without increasing cancer (annotated).pdf" Abstract A major goal in aging research is to improve health during aging. In the case of mice, genetic manipulations that shorten or lengthen telomeres result, respect- ively, in decreased or increased longevity. Based on this, we have tested the effects of a telomerase gene therapy in adult (1 year of age) and old (2 years of age) mice. Treatment of 1- and 2-year old mice with an adeno associated virus (AAV) of wide tropism expressing mouse TERT had remarkable bene?cial effects on health and ?tness, including insulin sensitivity, osteoporosis, neuromuscular coordination and several molecular biomarkers of aging. Importantly, telomer- ase-treated mice did not develop more cancer than their control littermates, suggesting that the known tumorigenic activity of telomerase is severely decreased when expressed in adult or old organisms using AAV vectors. Finally, telomerase-treated mice, both at 1-year and at 2-year of age, had an increase in median lifespan of 24 and 13%, respectively. These bene?cial effects were not observed with a catalytically inactive TERT, demonstrating that they require telomerase activity. Together, these results constitute a proof-of-principle of a role of TERT in delaying physiological aging and extending longevity in normal mice through a telomerase-based treatment, and demonstrate the feasibility of anti-aging gene therapy. Some notes -Spain paper -EMBO Molecular Medicine paper -q Treatment of 1- and 2-year old mice with an adeno associated virus (AAV) of wide tropism expressing mouse TERT had remarkable bene?cial effects on health and ?tness, including insulin sensitivity, osteoporosis, neuromuscular coordination and several molecular biomarkers of aging. -q Importantly, telomer- ase-treated mice did not develop more cancer than their control littermates, suggesting that the known tumorigenic activity of telomerase is severely decreased when expressed in adult or old organisms using AAV vectors. -q Finally, telomerase-treated mice, both at 1-year and at 2-year of age, had an increase in median lifespan of 24 and 13%, respectively. -demonstrate the feasibility of anti-aging gene therapy. -AAV9 mediated expression of mTERT leads to a stable and efficient systemic transduction of mouse tissues -mTERT ectopic expression late in life decreases the incidence of age-related osteoporosis and glucose intolerance -mTERT ectopic expression late in life improves scores in neuromuscular and object-recognition tests -A TERT-based gene therapy late in life significantly extends lifespan without increasing cancer -Telomerase expression late in life leads to overall telomere lengthening and decreased abundance of short telomeres in various tissues -AAV9-mTERT treatment late in life increases cyclinD1 R cells in mouse tissues -AAV9-mTERT treatment late in life improves metabolic and mitochondrial fitness -Delayed aging and increased longevity associated with increased TERT expression late in life requires of a catalytically active mTERT -q The gene therapy described here represents a novel type of therapeutic intervention against various age-related diseases. -about 15-20 mice in each group -Recombinant AAV vectors -Measurement of virus transduction efficiency -Telomere Q-FISH analysis on paraffin sections -Bone density (DEXA scan) -Skin measurements -Intraperitoneal glucose tolerance tests, insulin and IGF-1 -Neuromuscular coordination -TRAP assay -What was the cause of death of the treated mice? Why didn't they live longer? Telomerase reverse transcriptase synergizes with calorie restriction to increase health span and extend mouse longevity "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-07-2014d0952\Telomerase reverse transcriptase synergizes with calorie restriction to increase health span and extend mouse longevity.pdf" Abstract Caloric restriction (CR), a reduction of food intake while avoiding malnutrition, can delay the onset of cancer and age-related diseases in several species, including mice. In addition, depending of the genetic background, CR can also increase or decrease mouse longevity. This has highlighted the importance of identifying the molecular pathways that interplay with CR in modulating longevity. Significant lifespan extension in mice has been recently achieved through over-expression of the catalytic subunit of mouse telomerase (mTERT) in a cancer protective background. Given the CR cancer-protective effects in rodents, we set to address here whether CR impacts on telomere length and synergizes with mTERT to extend mouse longevity. CR significantly decreased tumor incidence in TERT transgenic (TgTERT) mice and extended their lifespan compared to wild-type (WT) controls under the same diet, indicating a synergy between TgTERT and CR in increasing mouse longevity. In addition, longitudinal telomere length measurements in peripheral blood leukocytes from individual mice showed that CR resulted in maintenance and/or elongation telomeres in a percentage of WT mice, a situation that mimics telomere dynamics in TgTERT cohorts. These results demonstrate that CR attenuates telomere erosion associated to aging and that synergizes with TERT over-expression in increasing “health span” and extending mouse longevity. Some notes -Bernardes de Jesus and Maria Blasco -Spain paper -PLOS one paper -q depending of the genetic background, CR can also increase or decrease mouse longevity -q CR significantly decreased tumor incidence in TERT transgenic (TgTERT) mice and extended their lifespan compared to wild-type (WT) controls under the same diet, indicating a synergy between TgTERT and CR in increasing mouse longevity -q These results demonstrate that CR attenuates telomere erosion associated to aging and that synergizes with TERT over-expression in increasing ‘‘health span’’ and extending mouse longevity -q At the metabolic level, CR results in improved insulin sensitivity and subsequent decrease in the fasting glucose, protecting from age-dependent metabolic syndrome and diabetes -q Calorie Restriction Leads to Significant Weight Loss in both WT and TgTERT Mice -q Stronger Effect of CR in Delaying Age-associated Pathologies in Mice Over-expressing TERT -q The Impact of CR Decreasing Molecular Markers of Aging is More Apparent in Mice Over-expressing TERT -Calorie Restriction Decreases the Rate of Telomere Shortening with Aging in Longitudinal Studies -Calorie Restriction Leads to Maintenance and/or Elongation of Telomeres in Individual Mice -Calorie Restriction Results in Longer Telomeres in Various Adult Mouse Tissues -Calorie Restriction Decreases Accumulation of Telomere- originated Chromosomal Aberrations with Aging -CR Synergizes with TERT Over-expression in Extending Mouse Longevity -In summary, CR is able to reduce the increased cancer incidence associated to TERT over-expression, which together with the increased ‘‘health span’’ associated to TERT over-expression could explain the synergistic effects of TERT and CR in increasing longevity -Caloric restriction increases median and maximum longevity and protects from cancer. -histopathology -DEXA -glucose tolerance tests -insulin, HOMA-IR, IGF1, and GH measurements -QFISH -quantification of phosphorylated H2AX -neuromuscular coordination test -what was the cr regime? --control mice: 92.5 kcal per week of chemically defined control diet --CR group had 74 kcal per week of chemically defined CR die for 2 weeks followed by 59.2 kcal per week of CR diet (AIN-93M 40% Restricted, Diet No. F05314, Bioserv). - -how many mice in each group? (18-20) -In particular, although TERT overexpressing mice presented a higher incidence of neoplasias than WT mice, the incidence of these neoplasias was reduced to a similar incidence than that of WT mice under CR Telomere Length and Telomerase Activity Impact the UV Sensitivity Syndrome Xeroderma Pigmentosum "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-08-2014d0943\Telomere Length and Telomerase Activity Impact the UV Sensitivity Syndrome Xeroderma Pigmentosum.pdf" Abstract Xeroderma pigmentosum (XP), a UV-sensitivity syndrome characterized by skin hyperpigmentation, premature aging, and increased skin cancer, is caused by defects in the nucleotide excision repair (NER) pathway. XP shares phenotypical characteristics with telomere-associated diseases like Dyskeratosis congenita and mouse models with dysfunctional telomeres, including mice deficient for telomerase (Terc-/- mice). Thus, we investigated a hypothesized role for telomerase and telomere dysfunction in the pathobiology of XP by comparing Xpc-/--mutant mice and Xpc-/-G1-G3Terc-/- double-mutant mice and exposed them to UV radiation. Chronically UV-exposed Xpc-/- skin displayed shorter telomeres on an average compared with wild-type skin. Strikingly, this effect was reversed by an additional deficiency in the telomerase. Moreover, aberrantly long telomeres were observed in the double-mutant mice. Telomere lengthening in the absence of telomerase suggested activation of the alternative lengthening of telomeres (ALT) in the UV-exposed skin of the double mutants. Mechanistic investigations revealed an elevated susceptibility for UV-induced p53 patches, known to represent precursor lesions of carcinomas, in Xpc-/-G1-G3Terc-/- mice where a high number of UV-induced skin tumors occurred that were characterized by aggressive growth. Taken together, our results establish a role for xeroderma pigmentosum, complementation group C (XPC) in telomere stability, particularly upon UV exposure. In absence of telomerase, critically short telomeres in XP mutants seem to aggravate this pathology, associated with an increased tumor incidence, by activating the ALT pathway of telomere lengthening Some notes -Cancer Research journal -CNIO paper -Gerdine Stout and Maria Blasco paper -corresponding author is Gerdine Stout -XP <- defects in the nucleotide excision repair (NER) pathway - Moreover, aberrantly long telomeres were observed in the double-mutant mice. Telomere lengthening in the absence of telomerase suggested activation of the alternative lengthening of telomeres (ALT) in the UV-exposed skin of the double mutants. --surprising result -q In absence of telomerase, critically short telomeres in XP mutants seem to aggravate this pathology, associated with an increased tumor incidence, by activating the ALT pathway of telomere lengthening. -knockout mice -UVB carcinogenesis -Q-FISH -cytogenetic analysis -ImmunoFISH -Xpc prevents telomere fragility -Longevity and aging in XpcTerc mouse model -UV-induced carcinogenesis is accelerated in XpcTerc mice -Chronic UV exposure leads to accelerated telomere shortening -Immunohistochemistry on UV-irradiated skin -Activation of ALT in UV-irradiated skin of Xpc / Terc  /  skinTelomere rejuvenation during nuclear reprogramming Telomere rejuvenation during nuclear reprogramming "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-01-2014d1112\Telomere rejuvenation during nuclear reprogramming.pdf" Abstract: Reprogramming of adult differentiated cells to a more pluripotent state has been achieved by various means, including somatic cell nuclear transfer (SCNT) and, more recently, by over expression of speciï¬?c transcription factors to generate the so-called induced pluripotent stem (iPS) cells. Since telomeres play an important role in the maintenance of chromosomal stability associated with continuous cell division, a key question for the quality of the resulting reprogrammed cells was to address whether nuclear reprogramming involves a full rejuvenation of telomeres. Recent work from our group and others demonstrate that telomeres are indeed rejuvenated during nuclear reprogramming. These ï¬?ndings also revealed that the structure of telomeric chromatin is dynamic and controlled by epigenetic programmes, which are reversed by reprogramming. some notes -telomeres are indeed rejuvenated during nuclear reprogramming. -review paper -Regulation of telomeres and telomerase -Telomeres and telomerase regulation during reprogramming by SCNT -SCNT: somatic cell nuclear transfer -The analysis of Dolly’s telomeres, cloned from a cultured mammary cell from a 6-year-old animal, revealed that they were shorter, by approximately 20%, when com- pared with age-matched controls -The demonstration of telomere elongation during SCNT opened new questions, -Telomeres and telomerase regulation during iPS cell generation -Telomerase activation is essential for the quality of the resulting iPS cells -Regulation of telomere reprogramming Telomere shortening and oxidative stress in aged macrophages results in impaired STAT5a phosphorylation "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-25-2014d1100\Telomere shortening and oxidative stress in aged macrophages results in impaired STAT5a phosphorylation.pdf"Telomere Shortening in Neural Stem Cells Disrupts Neuronal Differentiation and Neuritogenesis "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-30-2014d1027\Telomere Shortening in Neural Stem Cells Disrupts Neuronal Differentiation and Neuritogenesis.pdf" Abstract Proliferation in the subependymal zone (SEZ) and neurogenesis in the olfactory bulb decline in the forebrain of telomerase-deficient mice. The present work reveals additional effects of telomere shortening on neuronal differentiation, as adult multipotent progenitors withcriticallyshorttelomeresyieldreducednumbersofneuronsthat,furthermore,exhibitunderdevelopedneuriticarbors.Geneticdata indicate that the tumor suppressor protein p53 not only mediates the adverse effects of telomere attrition on proliferation and self- renewal but it is also involved in preventing normal neuronal differentiation of adult progenitors with dysfunctional telomeres. Inter- estingly, progenitor cells with short telomeres obtained from fetal brains do not exhibit any replicative defects but also fail to acquire a fully mature neuritic arbor, demonstrating cell cycle-independent effects of telomeres on neuronal differentiation. The negative effect of p53 on neuritogenesis is mechanistically linked to its cooperation with the Notch pathway in the upregulation of small GTPase RhoA kinases, Rock1 and Rock2, suggesting a potential link between DNA damage and the Notch signaling pathway in the control of neurito- genesis. We also show that telomerase expression is downregulated in the SEZ of aging mice leading to telomere length reductions in neurosphere-forming cells and deficient neurogenesis and neuritogenesis. Our results suggest that age-related deficits could be caused partly by dysfunctional telomeres and demonstrate that p53 is a central modulator of adult neurogenesis, regulating both the production and differentiation of postnatally generated olfactory neurons. notes and highlights -Our results suggest that age-related deficits could be caused partly by dysfunctional telomeres and demonstrate that p53 is a central modulator of adult neurogenesis, regulating both the production and differentiation of postnatally generated olfactory neurons. -Histological analysis -cell culture -cell transduction -immunohistochemistry -neuritic-dendritic measurements -Confocal -RNA isolation and semiquantitative and quantitative PCR analyses Telomere shortening relaxes X chromosome inactivation and forces global transcriptome alterations "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-30-2014d1027\Telomere shortening relaxes X chromosome inactivation and forces global transcriptome alterations.pdf" Abstract Telomeres are heterochromatic structures at chromosome ends es- sential for chromosomal stability. Telomere shortening and the ac- cumulation of dysfunctional telomeres are associated with organis- mal aging. Using telomerase-de?cient TRF2-overexpressing mice (K5TRF2/Terc / ) as a model for accelerated aging, we show that telomere shortening is paralleled by a gradual deregulation of the mammalian transcriptome leading to cumulative changes in a de?ned set of genes, including up-regulation of the mTOR and Akt survival pathways and down-regulation of cell cycle and DNA repair path- ways. Increased DNA damage from dysfunctional telomeres leads to reduced deposition of H3K27me3 onto the inactive X chromosome (Xi), impaired association of the Xi with telomeric transcript accumu- lations (Tacs), and reactivation of an X chromosome-linked K5TRF2 transgene that is subjected to X-chromosome inactivation in female mice with suf?ciently long telomeres. Exogenously induced DNA damage also disrupts Xi-Tacs, suggesting DNA damage at the origin of these alterations. Collectively, these ?ndings suggest that critically short telomeres activate a persistent DNA damage response that alters gene expression programs in a nonstochastic manner toward cell cycle arrest and activation of survival pathways, as well as impacts the maintenance of epigenetic memory and nuclear organization, thereby contributing to organismal aging. Telomere shortening relaxes X chromosome inactivation and forces global transcriptome alterations "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-30-2014d1027\Telomere shortening relaxes X chromosome inactivation and forces global transcriptome alterations.pdf" Some Notes -TRF2 overexpressing mice (K5TRF2/Terc-/-) -q Collectively, these ?ndings suggest that critically short telomeres activate a persistent DNA damage response that alters gene expression programs in a nonstochastic manner toward cell cycle arrest and activation of survival pathways, as well as impacts the maintenance of epigenetic memory and nuclear organization, thereby contributing to organismal aging. -histopathology and immunochemistry -telomere length analysis -TRF-based telomere length analysis -cell culture -gamma irradiation for DNA damage -western blot -northern blot analysis -RNA FISH -microarray analysisTelomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-31-2014d1002\Telomeres acquire embryonic stem cell characteristics in induced pluripotent stem cells.pdf" Abstract Telomere shortening is associated with organismal aging. iPS cells have been recently derived from old patients; however, it is not known whether telomere chromatin acquires the same characteristics as in ES cells. We show here that telomeres are elongated in iPS cells compared to the parental differentiated cells both when using four (Oct3/4, Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming factors and both from young and aged individuals. We demonstrate genetically that, during reprogram- ming, telomere elongation is usually mediated by telomerase and that iPS telomeres acquire the epige- netic marks of ES cells, including a low density of tri- methylated histones H3K9 and H4K20 and increased abundance of telomere transcripts. Finally, reprog- ramming ef?ciency of cells derived from increasing generations of telomerase-de?cient mice shows a dramatic decrease in iPS cell ef?ciency, a defect that is restored by telomerase reintroduction. Together, these results highlight the importance of telomere biology for iPS cell generation and function- ality. some notes q -We show here that telomeres are elongated in iPS cells compared to the parental differentiated cells both when using four (Oct3/4, Sox2, Klf4, cMyc) or three (Oct3/4, Sox2, Klf4) reprogramming factors and both from young and aged individuals. ^wow quite surprising and important -cMyc Is Dispensable for Telomerase Activation in Mouse iPS Cells -Telomere Elongation by Telomerase in iPS Cells -Impaired iPS Cell Generation from Late-Generation Terc / MEF with Critically Short Telomeres -Telomeric Heterochromatin in iPS Cells Acquires ES Cell Features -Increased Telomere Transcription in iPS Cells -Telomere Elongation in iPS Cells Derived from Old Donors -Generation of mouse iPS cells -TRF analysis -MEF: mouse embryonic fibroblast -Q-FISH analysis -B1-SINE Cobra Analysis for Global DNA Methylation -Analysis of Genomic Subtelomeric DNA Methylation -ChIP AssayTelomeres and telomerase in adult stem cells and pluripotent embryonic stem cells Marión RM, Blasco MA (2010). Telomeres and telomerase in adult stem cells and pluripotent embryonic stem cells. Adv Exp Med Biol 695, 118-131 I think this is from a book maybe. I can't seem to access it. http://link.springer.com/chapter/10.1007%2F978-1-4419-7037-4_9Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\Telomeric and extra-telomeric roles for telomerase and the telomere-binding proteins.pdf" Abstract Mammalian telomeres are formed by tandem repeats of the TTAGGG sequence, which are progressively lost with each round of cell division. Telomere protection requires a minimal length of TTAGGG repeats to allow the binding of shelterin, which prevents the activation of a DNA damage response (DDR) at chromosome ends. Telomere elongation is carried out by telomerase. Telomerase can also act as a transcriptional modulator of the Wnt–ß-catenin signalling pathway and has RNA-dependent RNA polymerase activity. Dysfunctional telomeres can lead to either cancer or ageing pathologies depending on the integrity of the DDR. This Review discusses the role of telomeric proteins in cancer and ageing through modulating telomere length and protection, as well as regulating gene expression by binding to non-telomeric sites. Some notes -CNIO paper -Nature reviews paper - q Telomere protection requires a minimal length of TTAGGG repeats to allow the binding of shelterin, which prevents the activation of a DNA damage response (DDR) at chromosome ends. --What is this minimal length? - q Telomerase can also act as a transcriptional modulator of the Wnt–ß-catenin signalling pathway and has RNA-dependent RNA polymerase activity -q when in a complex with the RNA component of mitochondrial RNA processing endoribonuclease (RMRP). --This is quite interesting that it is related to mitochondria -Telomerase: an end to the ‘end-replication problem’. -The telomeric chromatin. -nice diagram of a shelterin complex -telomere dysfunction and genomic instability -Oral leukoplakia -Chromatid dicentrics -Breakage–fusion–bridge cycles. -Human diseases linked to telomere dysfunction: Dyskeratosis congenita, Acquired aplastic anaemia , Idiopathic pulmonary fibrosis (IPF), Cartilage hair hypoplasia (CHH) -Defects in telomeric DNA replication -mouse models for telomerase -The susceptibility of telomeric DNA to genotoxic dam- age -q Telomeric DNA has an increased susceptibility to single-strand DNA damage that is induced by oxida- tive stress owing to the fact that guanine triplets are highly sensitive to oxidation 1 -Cell cycle control and endoreduplication. -extra-telomeric roles for a telomeric protein -the multitasking jobs of telomerase -telomerase and anticancer treatment -Telomerase recruitment to telomeres -q link telomeres with a plethora of biological processes, such as mitochondrial function, inflammation, embryonic development, regulation of gene expression, metabolism, stem cell homeostasis, adhesion and cancerTERRA transcripts are bound by a complex array of RNA-binding proteins some notes -nature communications article -I don't have access to it (I'll request it from interlibrary loan) "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-04-2014d0911\TERRA transcripts are bound by a complex array of RNA-binding proteins.pdf" Abstract Telomeres are transcribed from the telomeric C-rich strand, giving rise to UUAGGG repeat- containing telomeric transcripts or TERRA, which are novel structural components of telomeres. TERRA abundance is highly dependent on developmental status (including nuclear reprogramming), telomere length, cellular stresses, tumour stage and chromatin structure. However, the molecular mechanisms and factors controlling TERRA levels are still largely unknown. In this study, we identify a set of RNA-binding proteins, which endogenously bind and regulate TERRA in the context of primary mouse embryonic fibroblasts. The identification was carried out by biotin pull-down assays followed by LC-MALDI TOF/TOF mass spectrometry. Different members of the heterogeneous nuclear ribonucleoprotein family are among the ribonucleoprotein familythat bind more abundantly to TERRA. Downregulation ofTERRA—bound RBPs by small interfering RNA further shows that they can impact on TERRA abundance, their location and telomere lengthening. These findings anticipate an impact of TERRA—associated RBPs on telomere biology and telomeres diseases, such as cancer and aging. Some notes -In this study, we identify a set of RNA-binding proteins, which endogenously bind and regulate TERRA in the context of primary mouse embryonic fibroblasts -LC-MALDI TOF/TOF -biotin pull-down assays -Identiï¬?cation of TERRA-bound RBPs in primary MEFs -RBP = RNA- binding proteins -The hnRNPs bind to TERRA in vitro and in vivo. -TERRA-bound RBPs influence TERRA abundance and location. -Together, the results presented here show that TERRA tran- scripts are bound by a complex set of proteins, being hnRNP A1, A2B1, F and M the most abundant ones. -cell culture and siRNA transfection -protein identification by MS -western blot -immunoprecipitation -biotin pull-down -northern blot -mRNA stability -RNA-FISH -Q-FISH -polyadenylation assaysThe Hallmarks of Aging Leading Edge Review in Cell Journal 2013 Maria Blasco, Manuel Serrano, etc. -"F:\kurt\storage\CIM Research Folder\DR\2013\9-11-13\The Hallmarks of Aging.pdf" -"F:\kurt\storage\CIM Research Folder\DR\2013\9-11-13\The Hallmarks of Aging (annotated).pdf" The Hallmarks of Aging Leading Edge Review in Cell Journal 2013 Maria Blasco, Manuel Serrano, etc. -"F:\kurt\storage\CIM Research Folder\DR\2013\9-11-13\The Hallmarks of Aging.pdf" -"F:\kurt\storage\CIM Research Folder\DR\2013\9-11-13\The Hallmarks of Aging (annotated).pdf" Abstract Aging is characterized by a progressive loss of physiological integrity, leading to impaired function and increased vulnerability to death. This deterioration is the primary risk factor for major human pathologies, including cancer, diabetes, cardiovascular disorders, and neurodegenerative dis- eases. Aging research has experienced an unprecedented advance over recent years, particularly with the discovery that the rate of aging is controlled, at least to some extent, by genetic pathways and biochemical processes conserved in evolution. This Review enumerates nine tentative hall- marks that represent common denominators of aging in different organisms, with special emphasis on mammalian aging. These hallmarks are: genomic instability, telomere attrition, epigenetic alter- ations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication. A major challenge is to dissect the interconnectedness between the candidate hallmarks and their relative contributions to aging, with the ?nal goal of identifying pharmaceutical targets to improve human health during aging, with minimal side effects. Some notes -CNIO, Max Planck Institute Germany, London, France paper -corresponding author: Manuel Serrano -These hallmarks are: genomic instability, telomere attrition, epigenetic alter- ations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication -The cancer ?eld gained major momentum in 2000, with the publication of a landmark paper that enumerated six hallmarks of cancer (Hanahan and Weinberg, 2000), recently expanded to ten (Hanahan and Weinberg, 2011). T -At ?rst sight, cancer and aging may seem to be opposite pro- cesses: cancer is the consequence of an aberrant gain of cellular ?tness, whereas aging is characterized by a loss of ?tness. At a deeper level, however, cancer and aging share common origins. T -Therefore, cancer and aging can be regarded as two different manifestations of the same underlying process— namely, the accumulation of cellular damage. In addition, several of the pathologies associated with aging, such as atheroscle- rosis and in?ammation, involve uncontrolled cellular overgrowth or hyperactivity (Blagosklonny, 2008). B -ganisms (Gems and Partridge, 2013; Kenyon, 2010). Each hallmark should ideally ful?ll the following criteria: (1) it should manifest during normal aging; (2) its experimental aggravation should accelerate aging; and (3) its experimental amelioration should retard the normal aging pro- cess and hence increase healthy lifespan. Th -Genomic instability enomic Instability -One common denominator of aging is the accumulation of ge- netic damage throughout life - of DNA repair mech- anisms that are collectively capable of dealing with most of the damages in?icted to nuclear DNA -, defects in the nuclear architecture, known as laminopathies, can cause genome instability and result in premature aging syndromes -Nuclear DNA -asis. This is especially relevant when DNA dam- age impacts the functional competence of stem cells, thus compromising their role in tissue renewal -at de?ciencies in DNA repair mechanisms cause accelerated aging in mice and underlie several human progeroid syndromes, such as Werner syndrome, Bloom syndrome, xeroderma pigmentosum, trichothiodystro- phy, Cockayne syndrome, and Seckel syndrome (Gregg et al., 2012; Hoeijmakers, 2009; Murga et al., 2009). Moreover, trans- genic mice overexpressing BubR1, a mitotic checkpoint compo- nent that ensures accurate segregation of chromosomes, exhibit an increased protection against aneuploidy and cancer, as well as extended healthy lifespan (Baker et al., 2013) -Mitochondrial DNA -of mutant and wild-type genomes within the same cell, a phenom- enon that is referred to as ‘‘heteroplasmy.’’ -atory chain dysfunction in different tissues (Ameur et al., 2011). Studies of accelerated aging in HIV-infected patients treated with antiretroviral drugs, which interfere with mtDNA replication, have supported the concept of clonal expan- sion of mtDNA mutations originated early in life -nuclear architecture -ure Defects in the nuclear lamina can also cause genome instability -ity (Gonza- lez-Suarez et al., 2009; Liu et al., 2005). The nuclear lamina attracted the attention of aging researchers after the discovery that mutations in genes encoding protein components of this structure, or factors affecting their maturation and dynamics, cause accelerated aging syndromes such as the Hutchinson-Gil- ford and the Ne´ stor-Guillermo progeria syndromes (HGPS and NGPS, respectively) -g (Ragnauth et al., 2010; Scaf?di and Misteli, 2006). Telomere dysfunction also promotes progerin production in normal human ?broblasts upon prolonged in vitro culture, suggesting intimate links between telomere maintenance and progerin expression during normal aging -progerin is a truncated version of lamin A protein -. Restoration of the somatotrophic axis through hor- monal treatments or inhibition of NF- k B signaling also extends lifespan in these progeroid mice (Marin˜ o et al., 2010; Osorio et al., 2012). M -Telomere attrition -Indeed, ectopic expression of telomerase is suf?cient to confer immortality to otherwise mortal cells without causing oncogenic transforma- tion (Bodnar et al., 1998). I -c multiprotein complex known as shelterin -Telomerase de?ciency in humans is associated with prema- ture development of diseases, such as pulmonary ?brosis, dys- keratosis congenita, and aplastic anemia, which involve the loss of the regenerative capacity of different tissues (Armanios and Blackburn, 2012). T -Thus, mice with shortened or lengthened telomeres exhibit decreased or increased lifespans, respectively (Armanios et al., 2009; Blasco et al., 1997; Herrera et al., 1999; Rudolph et al., 1999; Toma´ s-Loba et al., 2008). R -e (Jaskelioff et al., 2011). Moreover, normal phys- iological aging can be delayed without increasing the incidence of cancer in adult wild-type mice by systemic viral transduction of telomerase (Bernardes de Jesus et al., 2012). -Epigenetic alterations -histone modifications -Deletion of components of histone methylation complexes (for H3K4 and for H3K27) extends longevity in nem- atodes and ?ies, respectively (Greer et al., 2010; Siebold et al., 2010). M -insulin/IGF-1 signaling pathway (Jin et al., 2011). I -Sir2, had a remarkable longevity activity (Guarente, 2011). O -findings on Sir-2.1 in question -on (Kan? et al., 2010; Kawahara et al., 2009; Zhong et al., 2010). Mutant mice that are de?cient in SIRT6 exhibit accelerated aging -n SIRT3 has been reported to mediate some of the bene?cial effects of dietary re- striction (DR) in longevity, though its effects are not due to his- tone modi?cations but, rather, due to the deacetylation of mito- chondrial proteins -s (Brown et al., 2013). Therefore, in mammals, at least three members of the sirtuin family—SIRT1, SIRT3 and SIRT6—contribute to healthy aging. -DNA Methylation -an age-associated global hypome- thylation -s (Pollina and Brunet, 2011) (see ‘‘Stem Cell Exhaustion’’). Nevertheless, there is no direct exper- imental demonstration thus far that organismal lifespan can be extended by altering patterns of DNA methylation. -chromatin remodeling -e NuRD complex, whose levels are dimin- ished in both normally and pathologically aged cells -in HP1 a have a shortened lifespan, whereas overexpression of this heterochromatin protein extends longevity in ?ies and delays the muscular deterioration characteristic of old age -s, epigenetic alterations can directly impinge on the regulation of telomere length, one of the hallmarks of aging. -trancriptional alterations -Aging is associated with an increase in transcriptional noise (Bahar et al., 2006) and an aberrant production and maturation of many mRNAs (Harries et al., 2011; Nicholas et al., 2010). M -r (Boulias and Horvitz, 2012; Toledano et al., 2012; Ugalde et al., 2011). Gain- and loss-of- function studies have con?rmed the capacity of several miRNAs to modulate longevity in Drosophila melanogaster and C. elegans -reversion of epigenetic changes -s Unlike DNA mutations, epigenetic alterations are—at least theo- retically—reversible, hence offering opportunities for the design of novel anti-aging treatments (Freije and Lo´ pez-Oti´n, 2012; Rando and Chang, 2012). Restoration of physiological H4 acet- ylation through administration of histone deacetylase inhibitors avoids the manifestation of age-associated memory impairment in mice --the efficacy of this histone deacetylase inhibitor treatment is surprising to me -t understanding and manipulating the epigenome holds promise for improving age-related pathologies and extending healthy lifespan. -loss of proteostasis -e regulators of age-related proteotoxicity, such as MOAG-4 -, chronic expression of unfolded, misfolded, or aggregated pro- teins contributes to the development of some age-related pa- thologies, such as Alzheimer’s disease, Parkinson’s disease, and cataracts -Chaperone-Mediated Protein Folding and Stability -r, transgenic worms and ?ies overexpressing chaperones are long-lived (Morrow et al., 2004; Walker and Lithgow, 2003). Also, mutant mice de?- cient in a cochaperone of the heat-shock family exhibit acceler- ated aging phenotypes -Small molecules may be also employed as pharmacological chaperones to assure the refolding of damaged proteins and to improve age-related phenotypes in model organisms (Calamini et al., 2012). --I wonder how it works that small molecules can be pharmacological chaperones -proteolytic systems -The activities of the two principal proteolytic systems implicated in protein quality control—namely, the autophagy-lysosomal system and the ubiquitin-proteasome system—decline with ag- ing (Rubinsztein et al., 2011; Tomaru et al., 2012), -Regarding autophagy, transgenic mice with an extra copy of the chaperone-mediated autophagy receptor LAMP2a do not experience aging-associated decline in autophagic activity and preserve improved hepatic function with aging (Zhang and Cuervo, 2008). Interventions using chemical inducers of macro- autophagy (another type of autophagy different than chaperone- mediated autophagy) have spurred extraordinary interest after the discovery that constant or intermittent administration of the mTOR inhibitor rapamycin can increase the lifespan of middle- aged mice (Blagosklonny, 2011; Harrison et al., 2009). N - other mechanisms, such as inhibition of the ribosomal S6 protein kinase 1 (S6K1) implicated in protein synthesis (Selman et al., 2009), could contribute to explain the longevity effects of rapamycin (see ‘‘Deregulated Nutrient Sensing’’). Spermidine, an -Dietary supplementation with u -6 polyunsaturated fatty acids also extends lifespan in nematodes through auto- phagy activation (O’Rourke et al., 2013). --I would think omega 6 does lots of other things too - increased expression of the protea- some subunit RPN-6 by the FOXO transcription factor DAF-16 confers proteotoxic stress resistance and extends lifespan in C. elegans (Vilchez et al., 2012). -lso remarkable examples of genetic manipulations that improve proteostasis and delay aging in mammals (Zhang and Cuervo, 2008). -Deregulated Nutrient Sensing -e somatotrophic axis - ‘‘insulin and IGF-1 signaling’’ (IIS) pathway. Remarkably, the IIS pathway is the most conserved aging-controlling pathway in evolution, and among its multiple targets are the FOXO family of transcription factors and the mTOR complexes, which are also involved in aging and conserved through evolution (Barzilai et al., 2012; Fontana et al., 2010; Kenyon, 2010). Genetic polymor- phismsor mutations that reduce the functions of GH, IGF-1 recep- tor, insulin receptor, or downstream intracellular effectors such as AKT, mTOR, and FOXO have been linked to longevity, both in hu- mans and in model organisms, further illustrating the major impact of trophic and bioenergetic pathways on longevity ( -g, dietary restriction (DR) increases lifespan or healthspan in all investigated eukaryote species, including nonhuman primates -The Insulin- and IGF-1-Signaling Pathway -rly, mild mitochondrial dam- age can induce a hormetic response (mitohormesis) that triggers adaptive compensatory processes. -ly, mice with increased dosage of the tumor suppressor PTEN have been reported to exhibit a general downmodulation of the IIS pathway and an increased energy expenditure that is associated with improved mitochondrial oxidative metabolism, as well as with an enhanced activity of the brown adipose tissue (Garcia-Cao et al., 2012; Ortega-Molina et al., 2012). -Other Nutrient-Sensing Systems: mTOR, AMPK, and Sirtuins -n: mTOR, for the sensing of high amino acid concentrations; AMPK, which senses low-energy states by detecting high AMP levels; and sir- tuins, which sense low-energy states by detecting high NAD + levels (Houtkooper et al., 2010) (Figure 4 -The mTOR kinase is part of two multiprotein complexes, mTORC1 and mTORC2, that regulate essentially all aspects of anabolic metabolism (Laplante and Sabatini, 2012). G -t mTOR inhibition phenocopies DR -Therefore, the downregulation of mTORC1/S6K1 appears as the critical mediator of mammalian longevity in rela- tion to mTOR. Mor -gh inhibition of TOR activity clearly has bene?cial effects during aging, it also has undesirable side effects, such as impaired wound healing, insulin resistance, cataracts, and testicular degeneration in mice (Wilkinso -mitochondrial dysfunction -As cells and organisms age, the ef?cacy of the respiratory chain tends to diminish, thus increasing electron leakage and reducing ATP generation (Green et al., 2011) (Figure 4B). Th -the mitochondrial free radical theory of aging (Hekimi et al., 2011). Of particular impact has been the unexpected observation that increased ROS may prolong lifespan in yeast and C. elegans (D -ed, genetic manipulations in mice that increase mitochondrial ROS and oxidative damage do not accelerate ag- ing -lly, genetic manipulations that impair mitochondrial function but do not increase ROS accel- erate aging -l. Beyond a certain threshold, ROS levels betray their original homeostatic purpose and eventually aggravate, rather than alleviate, the age-associated damage ( -e positive, negative, or neutral effects of ROS on aging. -mitochondrial integrity and biogenesis -subsequent p53-mediated repression of PGC-1 a and PGC-1 b (Sahin and DePinho, 2012). This mitochondrial decline also oc- curs during physiological aging in wild-type mice and can be partially reversed by telomerase activation (Be -SIRT3 may also directly control the rate of ROS production by deacety- lating manganese superoxide dismutase, a major mitochondrial antioxidant enzyme (Qiu et al., 2010; Tao et al., 2010). Co -Interestingly, endurance training and alternate-day fasting may improve healthspan through their capacity to avoid mito- chondrial degeneration (Castello et al., 2011; Safdar et al., 2011). I -, at least in part, through the induction of auto- phagy, for which both endurance training and fasting constitute potent triggers -mitohormesis - According to this concept, mild toxic treatments trigger bene?cial compensa- tory responses that surpass the repair of the triggering damage and actually produce an improvement in cellular ?tness when compared to the starting predamage conditions. Thu -hat metformin retards aging in worms by impairing folate and methionine metabolism of their intestinal microbiome ( -cellular senescence -This phenomenon was originally described by Hay?ick in human ?broblasts serially passaged in culture (Hay?ick and Moorhead, 1961). Today, we know that the senes- cence observed by Hay?ick is caused by telomere shortening (Bodnar et al., 1998), bu -nontelomeric DNA damage and derepression of the INK4/ARF locus, both of which progressively occur with chronological aging, are also capable of inducing senescence (Collado et al., 2007). T -e. Some studies have directly used senes- cence-associated b -galactosidase (SABG) to identify senes- cence in tissues -organisms -rs. In aged organisms, this turnover system may become inef?cient or may exhaust the regenerative capacity of progenitor cells -This proin?ammatory secretome may contribute to aging (see ‘‘Intercellular Communication’’). -The INK4a/ARF Locus and p53 -e. A recent account listed more than 50 oncogenic or mitogenic alterations that are able to induce senescence -ted p16 INK4a /Rb and p19 ARF /p53 path- ways remain, in general, the most important ones - (Krishnamurthy et al., 2004; Ressler et al., 2006). We are not aware of any other gene or protein whose expression is so robustly correlated with chronological aging across tissues, across species, and with a range of variation that, on average, is one order of magnitude between young and old tissues. -an 300 genome-wide association studies (GWAS) identi?ed the INK4a/ARF locus as the genomic locus that is genetically linked to the highest number of age-associated pathologies, including several types of cardiovascular diseases, diabetes, glaucoma, and Alzheimer’s disease -e INK4a/ARF locus as the best documented gene that controls human aging and aging-associated pathologies. -rk. A moderate enhancement of the senescence-inducing tumor suppressor pathways may extend longevity (Matheu et al., 2007, 2009), and at the same time, elimination of senescent cells in an exper- imental progeria model delays age-related pathologies (Baker et al., 2011). Therefore, two interventions that are conceptually opposite are able to extend healthspan. -The decline in the regenerative potential of tissues is one of the most obvious characteristics of aging (Figure 5B). F -Stem cell exhaustion - Telo- mere shortening is also an important cause of stem cell decline with aging in multiple tissues (Flores et al., 2005; Sharpless and DePinho, 2007). T -ch excessive proliferation leads to exhaustion and premature aging (Rera et al., 2011) -r, transplantation of muscle-derived stem cells from young mice to progeroid mice extends lifespan and improves degenerative changes of these animals even in tissues in which donor cells are not de- tected, suggesting that their therapeutic bene?t may derive from systemic effects caused by secreted factors -hat it is possible to rejuvenate human senescent cells by pharmacological inhibition of the GTPase CDC42, whose activity is increased in aged HSCs -at stem cell rejuvenation may reverse the aging phenotype at the organismal level -Altered intercellular communication -Thus, neurohormonal signaling (e.g., renin-angiotensin, adrenergic, insulin-IGF1 signaling) tends to be deregulated in aging as in?ammatory reactions increase, immunosurveillance against pathogens and premalignant cells declines, and -inflammation -A prominent aging-associated alteration in intercellular commu- nication is ‘‘in?ammaging,’’ i.e., a smoldering proin?ammatory phenotype that accompanies aging in mammals (Salminen et al., 2012). I -of obesity and type 2 diabetes, two condi- tions that contribute to and correlate with aging in the human population ( -er, one of the functions of the im- mune system is to recognize and eliminate senescent cells (see ‘‘Stem Cell Exhaustion’’), as well as hyperploid cells -nd conditional expression of an NF- k B inhibitor in the aged skin of transgenic mice causes the phenotypic rejuvenation of this tissue, as well as the restoration of the transcriptional signature corresponding to young age (Adler et al., 2007). L -. Consistently, GnRH treatment prevents aging-impaired neurogenesis and deceler- ates aging development in mice (Zhang et al., 2013). These ?nd- ings suggest that the hypothalamus may modulate systemic aging by integrating NF-kB-driven in?ammatory responses with GnRH-mediated neuroendocrine effects. -tor. Interestingly, in addition to directing in?ammatory cytokine mRNA decay, AUF1 contributes to maintaining telo- mere length by activating the expression of the telomerase cat- alytic subunit TERT (Pont et al., 2012), again demonstrating that one single factor may have a strong impact on different aging hallmarks - SIRT1 can downregulate in?ammation-related genes -other types of intercellular communication -e (Lefebvre et al., 2012). Conversely, lifespan-extending manipulations tar- geting one single tissue can retard the aging process in other tis- sues -me: There is nothing here about keeping the immune system on track and preventing autoimmunity with age (in fact the term autoimmunity is not even used) -me: There is nothing here about keeping cells healthy and happy through use and stimulation. I think that bored unused cells are unhealthy cells. -me: No mention of the term entropy (although the terms deregulated and noise are used) -ts (Conboy et al., 2005; Loffredo et al., 2013; Villeda et al., 2011). Moreover, the long-term administration of anti-in?amma- tory agents such as aspirin may increase longevity in mice and healthy aging in humans -ts, it appears possible to extend lifespan by manipu- lating the composition and functionahallmarkslity of the complex and dynamic intestinal bacterial ecosystem of the human body -l. Excitingly, proof of principle exists for rejuvenation through blood-borne systemic factors -y, molecular analysis of the genome-environment interac- tions that modulate aging will help to identify drug targets for longevity promotion (de Magalha˜ es et al., 2012)The load of short telomeres is increased and associated with lifetime number of depressive episodes in bipolar II disorder Abstract It has recently been hypothesized that bipolar disorders are associated with accelerated aging. Telomere dysfunction, a biomarker of aging, is determined by the load of short telomeres, rather than by the mean telomere length. To our knowledge, the load of short telomeres has not been reported in any psychiatric disorder. The aims of the study were to examine the load of short telomeres and the mean telomere length and their relationships with illness duration and lifetime number of depressive episodes in bipolar II disorder (BD-II). Some notes -paper in collobaroation with Oslo Norway -a biomarker of aging, is determined by the load of short telomeres, rather than by the mean telomere length - The load of short telomeres was significantly increased in patients with BD-II relative to healthy controls and may represent 13 years of accelerated aging -Depressive episode-related stress may accelerate telomere shortening and aging. -the load of short telomeres and mean telomere length were signi?cantly associated with lifetime number of depressive episodes, but not with illness duration The Rate of Increase of Short Telomeres Predicts Longevity in Mammals "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-06-2014d0918\The Rate of Increase of Short Telomeres Predicts Longevity in Mammals.pdf" Abstract Aberrantly short telomeres result in decreased longevity in both humans and mice with defective telomere maintenance. Normal populations of humans and mice present high interindividual variation in telomere length, but it is unknown whether this is associated with their lifespan potential. To address this issue, we performed a longitudinal telomere length study along the lifespan of wild-type and transgenic telomerase reverse transcriptase mice. We found that mouse telomeres shorten ~100 times faster than human telomeres. Importantly, the rate of increase in the percentage of short telomeres, rather than the rate of telomere shortening per month, was a significant predictor of lifespan in both mouse cohorts, and those individuals who showed a higher rate of increase in the percentage of short telomeres were also the ones with a shorter lifespan. These findings demonstrate that short telomeres have a direct impact on longevity in mammals, and they highlight the importance of performing longitudinal telomere studies to predict longevity. Some notes -CNIO paper -Cell reports paper -We found that mouse telomeres shorten apprx 100 times faster than human telomeres. -These ?nd- ings demonstrate that short telomeres have a direct impact on longevity in mammals, and they highlight the importance of performing longitudinal telomere studies to predict longevity. -q The Rate of Increase in the Per- centage of Short Telomeres and the Rate of Telomere Shortening Predicts Mouse Longevity -q Collectively, these ?ndings demonstrate that the rate of increase in the percentage of short telomeres during an individ- ual’s lifetime, rather than the rate of telomere shortening over time, determines longevity in mice. These results bring into ques- tion the prevailing viewpoint that telomere shortening does not in?uence replicative aging in WT mice -importance of longitudinal studies -histological analysis -HT-QFISHThe role of telomeres and telomerase in stem cell aging Abstract Stem cells regenerate our bodies. In a similar manner to match ignition, stem cell ‘‘ignition’’ has to be precisely tuned to avoid uncontrolled proliferation as may occur in tumors or, inversely, the lack of proliferation as happens in degenerative disorders. During the last years it has become evident that telomeres and telomerase are main components of the stem cell ‘‘ignition’’ mechanism, provid- ing a way to restrain cancer and delay aging. Some notes -Maintaining telomere homeostasis -Telomere length, telomerase and healthy life -Telomere shortening and stem cell aging - Sensing telomere damage in stem cell populations -Live fast, die young’’ vs. ‘‘Live slow, die old and, with help, very old -Delaying ageing avoiding telomere loss - The longest telomeres as a marker for adult stem cells -Telomerase, telomeres, and pluripotency TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice "C:\Users\kurtw_000\Box Sync\DocDR\2014\02-03-2014d1015\TPP1 is required for TERT recruitment, telomere elongation during nuclear reprogramming, and normal skin development in mice.pdf" Abstract The TPP1/ACD protein (hereafter TPP1) is a component of the shelterin complex at mammalian telomeres. Here we find that Tpp1-deficient mouse embryonic fibroblasts (MEFs) show increased chromosomal instability including sister chromatid fusions and chromosomes with multitelomeric signals related to telomere fragility. Tpp1 deletion decreases both TERT (the telomerase catalytic subunit) binding to telomeres in MEFs and telomerase function at chromosome ends in vivo. Abrogation of Tpp1 abolished net telomere elongation in the context of nuclear reprogramming of MEFs into induced pluripotent stem cells, whereas Tpp1 deletion in stratified epithelia of Tpp1?/?K5-Cre mice resulted in perinatal death, severe skin hyperpigmentation, and impaired hair follicle morphogenesis. p53 deficiency rescues skin hyperpigmentation and hair growth in these mice, indicating that p53 restricts proliferation of Tpp1-deficient cells. These results suggest a telomere-capping model where TPP1 protects telomere integrity and regulates telomerase recruitment to telomeres, thereby preventing early occurrence of degenerative pathologies. Some notes -Here we ?nd that Tpp1-de?cient mouse embryonic ?broblasts (MEFs) show increased chro- mosomal instability including sister chromatid fusions and chromosomes with multitelomeric sig- nals related to telomere fragility. -Tpp1-De?cient MEFs Undergo Rapid Proliferative Arrest Concomitant with DDR Activation at Chromosome Ends -Tpp1 D/D K5-Cre Mice Show Increased Telomere Damage and G2/M Mitotic Arrest in the Epidermis -TPP1 Prevents Sister Telomere Fusions and Telomere Fragility -TPP1 Is Required for TERT Binding to Telomeres and Telomere Maintenance In Vivo -TPP1 Is Required for Net Telomere Elongation during Nuclear Reprogramming -Tpp1-Conditional Deletion in Strati?ed Epithelia Leads to Perinatal Death, Skin Hyperpigmentation, and Severe Defects in Hair Follicle Morphogenesis -Severe Hair Morphogenesis Defects in Tpp1 D/D K5-Cre Epidermis -p53 De?ciency Rescues Skin Hyperpigmentation and Hair Growth Defects in Tpp1 D/D K5-Cre p53 / Mice - Instead, Tpp1 abrogation had a profound negative impact on hair follicle downgrowth, proliferation, and differentiation, hindering the establishment of a mature hair bulge SC compartment. Importantly, these defects are rescued by p53 abrogation, supporting a key role of p53 in mediating proliferative arrest in response to persistent telomere damage in vivoTRF1 controls telomere length and mitotic fidelity in epithelial homeostasis 01-26-2014d1627 "C:\Users\kurtw_000\Box Sync\DocDR\2014\01-26-2014d1626\TRF1 controls telomere length and mitotic fidelity in epithelial homeostasis.pdf" Abstract 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 check- point 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. notes and highlights some techniques used -real-time quantitative PCR -Q-FISH -culturing cells -TRF analysis -ChIP analysis -RNA dot blot -clonogenic assays -TRAP assay to measure telomerase activity -B1-SINE Cobra method to measure global DNA methylation -cytogenic analysis -mutant mouse strain K5TRF1 TRF1 is a stem cell marker and is essential for the generation of induced pluripotent stem cells Abstract TRF1 is a component of the shelterin complex that protects chromosome ends. TRF1 deficiency leads to early embryonic lethality and to severe organ atrophy when deleted in adult tissues. Here we generate a reporter mouse carrying a knock-in eGFP-TRF1 fusion allele to study the role of TRF1 in stem cell biology and tissue homeostasis. We find that eGFP-TRF1 expression in mice is maximal in known adult stem cell compartments and show that TRF1 ensures their functionality. eGFP-TRF1 is highly expressed in induced pluripotent stem cells, uncoupled from the telomere elongation associated with reprogramming. Selection of eGFP-TRF1-high induced pluripotent stem cells correlates with higher pluripotency as indicated by their ability to form teratomas and chimeras. We further show that TRF1 is necessary for both induction and maintenance of pluripotency, and that TRF1 is a direct transcriptional target of Oct3/4. Some notes -ASU doesn't have access to this article -Nature communications journal -CNIO paper -corresponding author: Maria Blasco