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- FoxM1 repression during human aging leads to mitotic decline and aneuploidy-driven full senescencePublication . Macedo, Joana Catarina; Vaz, Sara; Bakker, Bjorn; Ribeiro, Rui; Bakker, Petra Lammigje; Escandell, Jose Miguel; Ferreira, Miguel Godinho; Medema, René; Foijer, Floris; Logarinho, ElsaAneuploidy, an abnormal chromosome number, has been linked to aging and age-associated diseases, but the underlying molecular mechanisms remain unknown. Here we show, through direct live-cell imaging of young, middle-aged, and old-aged primary human dermal fibroblasts, that aneuploidy increases with aging due to general dysfunction of the mitotic machinery. Increased chromosome mis-segregation in elderly mitotic cells correlates with an early senescence-associated secretory phenotype (SASP) and repression of Forkhead box M1 (FoxM1), the transcription factor that drives G2/M gene expression. FoxM1 induction in elderly and Hutchison-Gilford progeria syndrome fibroblasts prevents aneuploidy and, importantly, ameliorates cellular aging phenotypes. Moreover, we show that senescent fibroblasts isolated from elderly donors' cultures are often aneuploid, and that aneuploidy is a key trigger into full senescence phenotypes. Based on this feedback loop between cellular aging and aneuploidy, we propose modulation of mitotic efficiency through FoxM1 as a potential strategy against aging and progeria syndromes.
- The fission yeast Stn1-Ten1 complex limits telomerase activity via its SUMO-interacting motif and promotes telomeres replicationPublication . Matmati, Samah; Vaurs, Mélina; Escandell, José M; Maestroni, Laetitia; Nakamura, Toru M; Ferreira, Miguel G; Géli, Vincent; Coulon, StéphaneMammalian CST (CTC1-STN1-TEN1) complex fulfills numerous functions including rescue of the stalled replication forks and termination of telomerase action. In fission yeast lacking the CTC1 ortholog, the Stn1-Ten1 complex restricts telomerase action via its sumoylation-mediated interaction with Tpz1TPP1. We identify a small ubiquitin-like modifier (SUMO)-interacting motif (SIM) in the carboxyl-terminal part of Stn1 and show that this domain is crucial for SUMO and Tpz1-SUMO interactions. Point mutations in the SIM (Stn1-226) lead to telomere elongation, impair Stn1-Ten1 recruitment to telomeres, and enhance telomerase binding, revealing that Stn1 SIM domain contributes to the inhibition of telomerase activity at chromosome ends. Our results suggest that Stn1-Ten1 promotes DNA synthesis at telomeres to limit single-strand DNA accumulation. We further demonstrate that Stn1 functions in the replication of telomeric and subtelomeric regions in a Taz1-independent manner. Genetic analysis reveals that misregulation of origin firing and/or telomerase inhibition circumvents the replication defects of the stn1-226 mutant. Together, our results show that the Stn1-Ten1 complex has a dual function at telomeres by limiting telomerase action and promoting chromosome end replication.
- Telomeres in aging and disease: lessons from zebrafishPublication . Carneiro, Madalena C.; de Castro, Inês Pimenta; Ferreira, Miguel GodinhoAge is the highest risk factor for some of the most prevalent human diseases, including cancer. Telomere shortening is thought to play a central role in the aging process in humans. The link between telomeres and aging is highlighted by the fact that genetic diseases causing telomerase deficiency are associated with premature aging and increased risk of cancer. For the last two decades, this link has been mostly investigated using mice that have long telomeres. However, zebrafish has recently emerged as a powerful and complementary model system to study telomere biology. Zebrafish possess human-like short telomeres that progressively decline with age, reaching lengths in old age that are observed when telomerase is mutated. The extensive characterization of its well-conserved molecular and cellular physiology makes this vertebrate an excellent model to unravel the underlying relationship between telomere shortening, tissue regeneration, aging and disease. In this Review, we explore the advantages of using zebrafish in telomere research and discuss the primary discoveries made in this model that have contributed to expanding our knowledge of how telomere attrition contributes to cellular senescence, organ dysfunction and disease.
- TERT promoter mutations in pancreatic endocrine tumours are rare and mainly found in tumours from patients with hereditary syndromesPublication . Vinagre, João; Nabais, Joana; Pinheiro, Jorge; Batista, Rui; Oliveira, Rui Caetano; Gonçalves, António Pedro; Pestana, Ana; Reis, Marta; Mesquita, Bárbara; Pinto, Vasco; Lyra, Joana; Cipriano, Maria Augusta; Ferreira, Miguel Godinho; Lopes, José Manuel; Sobrinho-Simões, Manuel; Soares, PaulaOne of the hallmarks of cancer is its unlimited replicative potential that needs a compensatory mechanism for the consequential telomere erosion. Telomerase promoter (TERTp) mutations were recently reported as a novel mechanism for telomerase re-activation/expression in order to maintain telomere length. Pancreatic endocrine tumors (PETs) were so far recognized to rely mainly on the alternative lengthening of telomeres (ALT) mechanism. It was our objective to study if TERTp mutations were present in pancreatic endocrine tumors (PET) and could represent an alternative mechanism to ALT. TERTp mutations were detected in 7% of the cases studied and were mainly associated to patients harbouring hereditary syndromes. In vitro, using PET-derived cell lines and by luciferase reporter assay, these mutations confer a 2 to 4-fold increase in telomerase transcription activity. These novel alterations are able to recruit ETS transcription factor members, in particular GABP-α and ETV1, to the newly generated binding sites. We report for the first time TERTp mutations in PETs and PET-derived cell lines. Additionally, our data indicate that these mutations serve as an alternative mechanism and in an exclusive manner to ALT, in particular in patients with hereditary syndromes.
- Short Telomeres in Key Tissues Initiate Local and Systemic Aging in ZebrafishPublication . Carneiro, Madalena C.; Henriques, Catarina M.; Nabais, Joana; Ferreira, Tânia; Carvalho, Tânia; Ferreira, Miguel GodinhoTelomeres shorten with each cell division and telomere dysfunction is a recognized hallmark of aging. Tissue proliferation is expected to dictate the rate at which telomeres shorten. We set out to test whether proliferative tissues age faster than non-proliferative due to telomere shortening during zebrafish aging. We performed a prospective study linking telomere length to tissue pathology and disease. Contrary to expectations, we show that telomeres shorten to critical lengths only in specific tissues and independently of their proliferation rate. Short telomeres accumulate in the gut but not in other highly proliferative tissues such as the blood and gonads. Notably, the muscle, a low proliferative tissue, accumulates short telomeres and DNA damage at the same rate as the gut. Together, our work shows that telomere shortening and DNA damage in key tissues triggers not only local dysfunction but also anticipates the onset of age-associated diseases in other tissues, including cancer.
- Telomerase Is Required for Zebrafish LifespanPublication . Henriques, Catarina M.; Carneiro, Madalena C.; Tenente, Inês M.; Jacinto, António; Ferreira, Miguel GodinhoTelomerase activity is restricted in humans. Consequentially, telomeres shorten in most cells throughout our lives. Telomere dysfunction in vertebrates has been primarily studied in inbred mice strains with very long telomeres that fail to deplete telomeric repeats during their lifetime. It is, therefore, unclear how telomere shortening regulates tissue homeostasis in vertebrates with naturally short telomeres. Zebrafish have restricted telomerase expression and human-like telomere length. Here we show that first-generation tert(-/-) zebrafish die prematurely with shorter telomeres. tert(-/-) fish develop degenerative phenotypes, including premature infertility, gastrointestinal atrophy, and sarcopaenia. tert(-/-) mutants have impaired cell proliferation, accumulation of DNA damage markers, and a p53 response leading to early apoptosis, followed by accumulation of senescent cells. Apoptosis is primarily observed in the proliferative niche and germ cells. Cell proliferation, but not apoptosis, is rescued in tp53(-/-)tert(-/-) mutants, underscoring p53 as mediator of telomerase deficiency and consequent telomere instability. Thus, telomerase is limiting for zebrafish lifespan, enabling the study of telomere shortening in naturally ageing individuals.
- Genome architecture is a selectable trait that can be maintained by antagonistic pleiotropyPublication . Teresa Avelar, Ana; Perfeito, Lília; Gordo, Isabel; Godinho Ferreira, MiguelChromosomal rearrangements are mutations contributing to both within and between species variation; however their contribution to fitness is yet to be measured. Here we show that chromosomal rearrangements are pervasive in natural isolates of Schizosaccharomyces pombe and contribute to reproductive isolation. To determine the fitness effects of chromosome structure, we constructed two inversions and eight translocations without changing the coding sequence. We show that chromosomal rearrangements contribute to both reproductive success in meiosis and growth rate in mitosis with a strong genotype by environment interaction. These changes are accompanied by alterations in gene expression. Strikingly, we find several examples leading to antagonistic pleiotropy. Even though chromosomal rearrangements may have a deleterious effect during sexual reproduction, some compensate with a strong growth advantage in mitosis. Our results constitute the first quantification of fitness effects caused by de novo mutations that result in chromosomal rearrangement variation and suggest a mechanism for their maintenance in natural populations.
- Spontaneous telomere to telomere fusions occur in unperturbed fission yeast cellsPublication . Almeida, H.; Godinho Ferreira, M.Telomeres protect eukaryotic chromosomes from illegitimate end-to-end fusions. When this function fails, dicentric chromosomes are formed, triggering breakage-fusion-bridge cycles and genome instability. How efficient is this protection mechanism in normal cells is not fully understood. We created a positive selection assay aimed at capturing chromosome-end fusions in Schizosaccharomyces pombe. We placed telomere sequences with a head to head arrangement in an intron of a selectable marker contained on a plasmid. By linearizing the plasmid between the telomere sequences, we generated a stable mini-chromosome that fails to express the reporter gene. Whenever the ends of the mini-chromosome join, the marker gene is reconstituted and fusions are captured by direct selection. Using telomerase mutants, we recovered several fusion events that lacked telomere sequences. The end-joining reaction involved specific homologous subtelomeric sequences capable of forming hairpins, suggestive of ssDNA stabilization prior to fusing. These events occurred via microhomology-mediated end-joining (MMEJ)/single-strand annealing (SSA) repair and also required MRN/Ctp1. Strikingly, we were able to capture spontaneous telomere-to-telomere fusions in unperturbed cells. Similar to disruption of the telomere regulator Taz1/TRF2, end-joining reactions occurred via non-homologous end-joining (NHEJ) repair. Thus, telomeres undergo fusions prior to becoming critically short, possibly through transient deprotection. These dysfunction events induce chromosome instability and may underlie early tumourigenesis.
- Cdk1 Restrains NHEJ through Phosphorylation of XRCC4-like Factor Xlf1Publication . Hentges, Pierre; Waller, Helen; Reis, Clara C.; Ferreira, Miguel Godinho; Doherty, Aidan J.Eukaryotic cells use two principal mechanisms for repairing DNA double-strand breaks (DSBs): homologous recombination (HR) and nonhomologous end-joining (NHEJ). DSB repair pathway choice is strongly regulated during the cell cycle. Cyclin-dependent kinase 1 (Cdk1) activates HR by phosphorylation of key recombination factors. However, a mechanism for regulating the NHEJ pathway has not been established. Here, we report that Xlf1, a fission yeast XLF ortholog, is a key regulator of NHEJ activity in the cell cycle. We show that Cdk1 phosphorylates residues in the C terminus of Xlf1 over the course of the cell cycle. Mutation of these residues leads to the loss of Cdk1 phosphorylation, resulting in elevated levels of NHEJ repair in vivo. Together, these data establish that Xlf1 phosphorylation by Cdc2(Cdk1) provides a molecular mechanism for downregulation of NHEJ in fission yeast and indicates that XLF is a key regulator of end-joining processes in eukaryotic organisms.
- Positive Epistasis Drives the Acquisition of Multidrug ResistancePublication . Trindade, S.; Sousa, A.; Xavier, KB.; Dionisio, F.; Ferreira, MG.; Gordo, I.The evolution of multiple antibiotic resistance is an increasing global problem. Resistance mutations are known to impair fitness, and the evolution of resistance to multiple drugs depends both on their costs individually and on how they interact-epistasis. Information on the level of epistasis between antibiotic resistance mutations is of key importance to understanding epistasis amongst deleterious alleles, a key theoretical question, and to improving public health measures. Here we show that in an antibiotic-free environment the cost of multiple resistance is smaller than expected, a signature of pervasive positive epistasis among alleles that confer resistance to antibiotics. Competition assays reveal that the cost of resistance to a given antibiotic is dependent on the presence of resistance alleles for other antibiotics.