Telomere biology of trypanosomatids: beginning to answer some questions

Studies of telomere structure and maintenance in trypanosomatids have provided insights into the evolutionary origin and conservation of some telomeric components shared by trypanosomes and vertebrates. For example, trypanosomatid telomeres are maintained by telomerase and consist of the canonical TTAGGG repeats, which in Trypanosoma brucei can form telomeric loops (t-loops). However, the telomeric chromatin of trypanosomatids is composed of organ ism-specific proteins and other proteins that share little sequence similarity with their vertebrate counterparts. Because telomere maintenance mechanisms are essential for genome stability, we propose that the particular features shown by the trypanosome telomeric chromatin hold the key for the design of antiparasitic drugs.

Embryonic exposure to corticosterone modifies the juvenile stress response, oxidative stress, and telomere length.

Early embryonic exposure to maternal glucocorticoids can broadly impact physiology and behaviour across phylogenetically diverse taxa. The transfer of maternal glucocorticoids to offspring may be an inevitable cost associated with poor environmental conditions, or serve as a maternal effect that alters offspring phenotype in preparation for a stressful environment. Regardless, maternal glucocorticoids are likely to have both costs and benefits that are paid and collected over different developmental time periods. We manipulated yolk corticosterone (cort) in domestic chickens (Gallus domesticus) to examine the potential impacts of embryonic exposure to maternal stress on the juvenile stress response and cellular ageing. Here, we report that juveniles exposed to experimentally increased cort in ovo had a protracted decline in cort during the recovery phase of the stress response. All birds, regardless of treatment group, shifted to oxidative stress during an acute stress response. In addition, embryonic exposure to cort resulted in higher levels of reactive oxygen metabolites and an over-representation of short telomeres compared with the control birds. In many species, individuals with higher levels of oxidative stress and shorter telomeres have the poorest survival prospects. Given this, long-term costs of glucocorticoid-induced phenotypes may include accelerated ageing and increased mortality.

Telomere length is associated with disease severity and declines with age in dyskeratosis congenita

Dyskeratosis congenita is a cancer-prone bone marrow failure syndrome caused by aberrations in telomere biology.

TINF2, a component of the shelterin telomere protection complex, is mutated in dyskeratosis congenita

Patients with dyskeratosis congenita (DC), a heterogeneous inherited bone marrow failure syndrome, have abnormalities in telomere biology, including very short telomeres and germline mutations in DKC1, TERC, TERT, or NOP10, but approximately 60% of DC patients lack an identifiable mutation. With the very short telomere phenotype and a highly penetrant, rare disease model, a linkage scan was performed on a family with autosomal-dominant DC and no mutations in DKCI, TERC, or TERT. Evidence favoring linkage was found at 2p24 and 14q11.2, and this led to the identification of TINF2 (14q11.2) mutations, K280E, in the proband and her five affected relatives and TINF2 R282H in three additional unrelated DC probands, including one with Revesz syndrome; a fifth DC proband had a R282S mutation. TINF2 mutations were not present in unaffected relatives, DC probands with mutations in DKC1, TERC, or TERT or 298 control subjects. We demonstrate that a fifth gene, TINF2, is mutated in classical DC and, for the first time, in Revesz syndrome. This represents the first shelterin complex mutation linked to human disease and confirms the role of very short telomeres as a diagnostic test for DC.

The Putative Leishmania Telomerase Rna (leishter) Undergoes Trans-splicing And Contains A Conserved Template Sequence.

Telomerase RNAs (TERs) are highly divergent between species, varying in size and sequence composition. Here, we identify a candidate for the telomerase RNA component of Leishmania genus, which includes species that cause leishmaniasis, a neglected tropical disease. Merging a thorough computational screening combined with RNA-seq evidence, we mapped a non-coding RNA gene localized in a syntenic locus on chromosome 25 of five Leishmania species that shares partial synteny with both Trypanosoma brucei TER locus and a putative TER candidate-containing locus of Crithidia fasciculata. Using target-driven molecular biology approaches, we detected a ∼2,100 nt transcript (LeishTER) that contains a 5' spliced leader (SL) cap, a putative 3' polyA tail and a predicted C/D box snoRNA domain. LeishTER is expressed at similar levels in the logarithmic and stationary growth phases of promastigote forms. A 5'SL capped LeishTER co-immunoprecipitated and co-localized with the telomerase protein component (TERT) in a cell cycle-dependent manner. Prediction of its secondary structure strongly suggests the existence of a bona fide single-stranded template sequence and a conserved C[U/C]GUCA motif-containing helix II, representing the template boundary element. This study paves the way for further investigations on the biogenesis of parasite TERT ribonucleoproteins (RNPs) and its role in parasite telomere biology.

Distinct tumor suppressor mechanisms evolve in rodent species that differ in size and lifespan

Large, long-lived species experience more lifetime cell divisions and hence a greater risk of spontaneous tumor formation than smaller, short-lived species. Large, long-lived species are thus expected to evolve more elaborate tumor suppressor systems. In previous work, we showed that telomerase activity coevolves with body mass, but not lifespan, in rodents: telomerase activity is repressed in the somatic tissues of large rodent species but remains active in small ones. Without telomerase activity, the telomeres of replicating cells become progressively shorter until, at some critical length, cells stop dividing. Our findings therefore suggested that repression of telomerase activity mitigates the increased risk of cancer in larger-bodied species but not necessarily longer-lived ones. These findings imply that other tumor suppressor mechanisms must mitigate increased cancer risk in long-lived species. Here, we examined the proliferation of fibroblasts from 15 rodent species with diverse body sizes and lifespans. We show that, consistent with repressed telomerase activity, fibroblasts from large rodents undergo replicative senescence accompanied by telomere shortening and overexpression of p16(Ink4a) and p21(Cip1/Waf1) cycline-dependent kinase inhibitors. Interestingly, small rodents with different lifespans show a striking difference: cells from small shorter-lived species display continuous rapid proliferation, whereas cells from small long-lived species display continuous slow proliferation. We hypothesize that cells of small long-lived rodents, lacking replicative senescence, have evolved alternative tumor-suppressor mechanisms that prevent inappropriate cell division in vivo and slow cell growth in vitro. Thus, large-bodied species and small but long-lived species have evolved distinct tumor suppressor mechanisms.

Studying the molecular causes of ageing in ants

RÉSUMÉ DE THÈSE Au cours de ma thèse, je me suis intéressée aux causes physiologiques du vieillissement en utilisant les fourmis comme modèle. Les trois castes de fourmis - les mâles, les ouvrières et les reines - présentent des longévités très différentes, tout en étant génétiquement identiques. Ceci implique que les différences de longévité sont dues à des variations entre castes dans le pattern d'expression de gènes. Mon travail chez la fourmi a consisté d'une part à mettre en place les outils pour identifier de tels gènes à grande échelle, de l'autre à étudier le rôle de gènes et de mécanismes qui affectent la longévité chez d'autres espèces. Pour identifier de nouveaux gènes potentiellement impliqués dans le vieillissement, nous avons développé des puces à ADN. Cette technique permet la comparaison du niveau d'expression de milliers de gènes entre deux échantillons. L'application de cette méthode aux reines et ouvrières adultes nous a jusqu'à présent permis d'identifier neuf gènes surexprimés chez les reines. Trois d'entre eux sont potentiellement impliqués dans le maintien et la réparation du soma, deux processus qui sont supposés avoir un impact crucial sur la longévité. Parmi les mécanismes impliqués dans le vieillissement chez d'autres espèces, nous nous sommes principalement intéressés aux télomères, qui sont les extrémités des chromosomes. Chez les vertébrés, les télomères se raccourcissent à chaque division cellulaire, entre autres parce que l'ADN polymérase ne peut répliquer cette partie des chromosomes en entier. Or des télomères courts entravent la prolifération des cellules et peuvent même induire l'apoptose, ce qui pourrait se répercuter sur la capacité des organismes à régénérer des tissus. J'ai pu montrer que chez les fourmis mâles (la caste qui vit le moins longtemps) les télomères se raccourcissent beaucoup plus vite que chez les reines et les ouvrières. L'explication la plus plausible pour cette différence est que les mâles, étant adapté à une vie très éphémère, n'investissent qu'un minimum d'énergie dans la machinerie de maintenance qui assure le bon fonctionnement des cellules. Ces résultats sont intéressants car ils permettent pour la première fois de faire le lien entre les théories évolutives du vieillissement et la biologie des télomères. THESIS ABSTRACT During my thesis I used ants as a model to study the proximate (i.e., molecular) causes of ageing and lifespan determination. Ant queens, workers and males differ tremendously in lifespan, although all three castes are genetically identical. Importantly, this implies that genes and molecular pathways responsible for modulating lifespan are regulated in a caste-specific manner. To find new genes potentially involved in ageing, we first constructed 371-gene-cDNA microarrays for the ant L. niger. This molecular tool can be used to survey the relative gene expression levels of two samples for thousands of genes simultaneously. By applying this method to adult queens and workers we identified nine genes that are overexpressed in queens. Three of them are putatively involved in somatic maintenance and repair, two processes that have been previously suggested as important for ageing and lifespan determination. We expect to identify many more candidate genes in the near future by using the 9000-gene fire ant microarrays we have recently developed. We also investigated whether factors linked to ageing in other organisms could affect lifespan determination in ants. One project was on telomeres, the ends of linear chromosomes. For various reasons telomeres shorten with every cell division. Since short telomeres can lead to cellular defects such as impaired cell division, telomeres have been hypothesized as playing a role in ageing. We tested whether telomere length in ant somatic tissues correlates with caste-specific lifespan in young adults. The short-lived L. niger mates did indeed have significantly shorter telomeres than the longer-lived queens and workers, probably because telomere attrition is faster in males than in queens and workers. Queens did not, however, have longer telomeres than the shorter-lived workers. These findings are consistent with the idea that telomere length may play a role in ageing under some circumstances, but they also clearly demonstrate that other factors must be involved. We argue that sex-specific telomere length patterns in ants ultimately reflect adaptive differences in the level of somatic maintenance between males and females, and thus create a link between telomere biology and the evolutionary theory of ageing.

Verificação da formação de complexos protéicos nos telômeros de L. amazonensis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

The putative Leishmania telomerase RNA (leishTER) undergoes trans-splicing and contains a conserved template sequence

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Telomeres and prognosis in patients with chronic lymphocytic leukaemia

In the present study, telomere length, telomerase activity, the mutation load of immunoglobulin variable heavy chain (IGHV) genes, and established prognostic factors were investigated in 78 patients with chronic lymphocytic leukaemia (CLL) to determine the impact of telomere biology on the pathogenesis of CLL. Telomere length was measured by an automated multi-colour flow-FISH, and an age-independent delta telomere length ( TL) was calculated. CLL with unmutated IGHV genes was associated with shorter telomeres (p = 0.002). Furthermore, we observed a linear correlation between the frequency of IGHV gene mutations and elongation of telomeres (r = 0.509, p < 0.001). With respect to prognosis, a threshold TL of -4.2 kb was the best predictor for progression-free and overall survival. TL was not significantly altered over time or with therapy. The correlation between the mutational load in IGHV genes and the TL in CLL might reflect the initial telomere length of the putative cell of origin (pre- versus post-germinal center B cells). In conclusion, the TL is a reliable prognostic marker for patients with CLL. Short telomeres and high telomerase activity as occurs in some patients with CLL with a worse prognosis might be an ideal target for treatment with telomerase inhibitors.

Telomeres: Linking stress and survival, ecology and evolution.

Telomeres are protective structures at the ends of eukaryotic chromosomes. The loss of telomeres through cell division and oxidative stress is related to cellular aging, organismal growth and disease. In this way, telomeres link molecular and cellular mechanisms with organismal processes, and may explain variation in a number of important life-history traits. Here, we discuss how telomere biology relates to the study of physiological ecology and life history evolution. We emphasize current knowledge on how telomeres may relate to growth, survival and lifespan in natural populations. We finish by examining interesting new connections between telomeres and the glucocorticoid stress response. Glucocorticoids are often employed as indices of physiological condition, and there is evidence that the glucocorticoid stress response is adaptive. We suggest that one way that glucocorticoids impact organismal survival is through elevated oxidative stress and telomere loss. Future work needs to establish and explore the link between the glucocorticoid stress response and telomere shortening in natural populations. If a link is found, it provides an explanatory mechanism by which environmental perturbation impacts life history trajectories.

Short telomeres and high telomerase activity in T-cell prolymphocytic leukemia

To test the role of telomere biology in T-cell prolymphocytic leukemia (T-PLL), a rare aggressive disease characterized by the expansion of a T-cell clone derived from immuno-competent post-thymic T-lymphocytes, we analyzed telomere length and telomerase activity in subsets of peripheral blood leukocytes from 11 newly diagnosed or relapsed patients with sporadic T-PLL. Telomere length values of the leukemic T cells (mean+/-s.d.: 1.53+/-0.65 kb) were all below the 1st percentile of telomere length values observed in T cells from healthy age-matched controls whereas telomere length of normal T- and B cells fell between the 1st and 99th percentile of the normal distribution. Leukemic T cells exhibited high levels of telomerase and were sensitive to the telomerase inhibitor BIBR1532 at doses that showed no effect on normal, unstimulated T cells. Targeting the short telomeres and telomerase activity in T-PLL seems an attractive strategy for the future treatment of this devastating disease.

Telomerase expression in chickens: Constitutive activity in somatic tissues and down-regulation in culture

Although human and rodent telomeres have been studied extensively, very little is known about telomere dynamics in other vertebrates. Moreover, our current dependence on mice as a model for human tumorigenesis and aging poses a problem because human and mouse telomere biology is very different. To explore whether chickens might provide a more useful model, we have examined telomerase activity and telomere length in chicken tissues as well as in primary cell cultures. Although chicken telomeres resemble human telomeres in that they are 8–20 kb in length, the distribution of telomerase activity in chickens resembles what is found in mice. Active enzyme is present in germline tissue as well as in a wide range of somatic tissues. Because chicken cells exhibit extremely low rates of spontaneous immortalization, this finding indicates that constitutive telomerase expression does not necessarily lead to an increased immortalization frequency. Finally, we found that telomerase activity is greatly down-regulated when primary cultures are established from chicken embryos. Although this down-regulation explains the telomere loss and replicative senescence that we observed in fibroblast cultures, it raises questions concerning how relevant studies of senescence in primary cell cultures are to aging in whole animals.

Telomeres in aging and disease: lessons from zebrafish

Age 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.

Criblage génétique à la recherche de nouveaux gènes essentiels influençant l’homéostasie des télomères chez Saccharomyces cerevisiae : Un défi de tailles.

Résumé : Chez la levure Saccharomyces cerevisiae, la régulation de la longueur des télomères témoigne de la compensation entre mécanismes d'érosion (exonucléases, réplication semi-conservative et résection), facteurs d’élongation (la télomérase, transcriptase inverse à l'action retrouvée dans 90% des cancers humains) et actions de diverses protéines de régulation télomérique spécifiques, conférant aux télomères leur caractère de « capuchon » protégeant les extrémités des chromosomes eucaryotes. Afin de savoir si les gènes impossibles à déléter, car essentiels à la survie cellulaire, jouent aussi un rôle sur l’homéostasie télomérique, j'ai réalisé un criblage génétique utilisant des mutants tet-off de la levure pour lesquels la sous-expression considérable d'un gène essentiel a été induite de façon conditionnelle. Ceci permet d’étudier les effets qui en résultent sur l’homéostasie des télomères. Au total, mon travail a traité plus de 662 gènes essentiels pour lesquels j'ai analysé le phénotype de longueur des télomères de manière qualitative par comparaison des télomères de souches mutées par rapport à ceux de souches de type sauvage. Puis, grâce à l’amélioration technique que j'ai mise au point, la quantification de la taille des répétitions télomériques de 300 de ces souches a déjà pu être précisément analysée. Il est notable que tous les gènes essentiels étudiés ici ont des effets très différents qui résultent en des chromosomes possédant des télomères de longueur très inégale. Pour près de 40% des mutants analysés, les tailles de télomères sont apparues critiquement différentes de celles normalement présentées par la levure, beaucoup de ces gènes essentiels étant impliqués dans des mécanismes affectant le cycle cellulaire, la réparation, etc. La majorité des gènes criblés apporte un important complément d’information dans une littérature presque inexistante sur les effets de gènes essentiels de la levure au niveau de la biologie des télomères. C’est le cas des mutations de YHR122W (montrant des télomères long) et YOR262W (télomères courts), deux gènes qui sont apparus d'après mes résultats nécessaires au maintien de l'homéostasie télomérique (prenant place dans un grand ensemble de gènes que j’ai dénommé gènes ETL pour Essential for Telomere Length Maintenance).