Functional Analysis of the Human Telomeric Protein TRF2

TTAGGG Repeat Binding Factor 2 (TRF2) is a ubiquitous human telomeric protein present at all telomeres throughout the cell cycle. TRF2 has been proposed remodel telomeres into large lasso-like structures named t-loops. Removal of TRF2 from telomeres by expression of a dominant negative allele of TRF2 (TRF2ABAM) led dissolution of these structures and appearance of end-to-end fusions visible in metaphase and anaphase cells. Fusion formation was accompanied by the loss of Goverhang, but not by the loss of double stranded telomeric DNA, which can be documented to persist at the sites of fusions. Fusions were covalent and their formation was dependent on the nonhomologous end-joining (NHEJ) pathway as evidenced by the failure to form fusions in cells lacking Ligase IV. Furthermore, the fusions initiated genomic instability. Expression of TRF2ABAM in primary human cells resulted in induction of senescence. The arrested cells exhibited elevated levels of p53, stabilization of was accompanied by induction of its downstream effectors p21 and Bax. The Rb pathway was also affected, with pRb becoming hypophosphorylated. The importance of both the p53 and the Rb pathways was further evident from the fact that the expression of SV40 Tag alone bypassed the growth arrest. Separate elimination of p53 or Rb function could not do so, and the activity of the ATM PI3 kinase was not necessary the arrest. Expression of a different truncation allele of TRF2, TRF2AB also resulted in senescence but the growth arrest was accompanied by rapid loss of telomeric DNA. In addition, this allele induced an unanticipated chromosome breakage phenotype. Long-term overexpression of full length T R F 2 resulted in the gradual shortening of telomeres, suggesting that TRF2 is a regulator of telomere maintenance. Although it is clear that TRF2 acts through positively regulating the shortening activities at telomeres, it is unknown at this time whether it influences the telomerase pathway. Besides being present at telomeres, TRF2 also appears to be localized to centrosomes. The functional consequences of such localization are not known at this time; however, it is shown that telomere dysfunction can result in genome reduplication

Attitudes of students toward people with disabilities, moral identity and inclusive education : a two-level analysis

Background : attitudes toward people with disabilities are a frequent subject of studies. However, there are few complex studies with personal explanatory variables. Thus, in our study we have conducted an analysis at both the individual and classroom level, as well as we have examined between-levels interactions. Methods: 1525 students without disabilities participated in the cross-sectional study, in which we analyzed attitudes toward people with disabilities, and moral identity in traditional and in inclusive classroom settings. Results: the results show that individual and classroom moral identity, as well as learning in an inclusive classroom, predict a reduction of negative attitudes toward people with disabilities. Moreover, we have reported some interesting interactions between these two levels. Conclusions: the results obtained are important for educational practice

Theory of mind goes to school : does educational environment influence the development of theory of mind in middle childhood?

Previous research has shown that the development of theory of mind (ToM) depends on various individual and social factors, but very little research has examined the role of the natural educational environment in the development of ToM in middle childhood. In accordance with the importance of social factors in development, in this longitudinal study of 156 typically developing children, we investigated whether educational setting - classes containing children with disabilities (inclusive) or without such children (general education) - is associated with enhanced ToM development. ToM was measured with the ToM Scale, the Chocolate task and the Faux Pas Recognition Test. Analysis showed that ToM development was better among children educated in inclusive classes than among those educated in traditional classes. The results have implications for ToM development among children with and without disabilities as well as for educational practice

Examining the effectiveness of naturalistic social skills training in developing social skills and theory of mind in preschoolers with ASD

We compared the effectiveness of two programs for developing social skills, ‘Play Time/Social Time’ (PT/ST) and ‘I Can Problem Solve’ (ICPS), in improving the social skills and theory of mind (ToM) of preschoolers with ASD. The experiment took place in a classroom setting. Fifty-two children attended and data were analyzed with latent growth curve models. Comparison with a control group indicated that both programs were effective in developing social skills. The PT/ST program was more effective than ICPS in developing interaction skills; both programs improved children’s ability to cope with difficult social situations. The ICPS program was marginally effective in developing ToM when compared with PT/ST and control condition. These results are relevant to children with ASD and their teachers

The Telomeric Protein TRF2 Binds the ATM Kinase and Can Inhibit the ATM-Dependent DNA Damage Response

The telomeric protein TRF2 is required to prevent mammalian telomeres from activating DNA damage checkpoints. Here we show that overexpression of TRF2 affects the response of the ATM kinase to DNA damage. Overexpression of TRF2 abrogated the cell cycle arrest after ionizing radiation and diminished several other readouts of the DNA damage response, including phosphorylation of Nbs1, induction of p53, and upregulation of p53 targets. TRF2 inhibited autophosphorylation of ATM on S1981, an early step in the activation of this kinase. A region of ATM containing S1981 was found to directly interact with TRF2 in vitro, and ATM immunoprecipitates contained TRF2. We propose that TRF2 has the ability to inhibit ATM activation at telomeres. Because TRF2 is abundant at chromosome ends but not elsewhere in the nucleus, this mechanism of checkpoint control could specifically block a DNA damage response at telomeres without affecting the surveillance of chromosome internal damage

Sister telomeres rendered dysfunctional by persistent cohesion are fused by NHEJ

Telomeres protect chromosome ends from being viewed as double-strand breaks and from eliciting a DNA damage response. Deprotection of chromosome ends occurs when telomeres become critically short because of replicative attrition or inhibition of TRF2. In this study, we report a novel form of deprotection that occurs exclusively after DNA replication in S/G2 phase of the cell cycle. In cells deficient in the telomeric poly(adenosine diphosphate ribose) polymerase tankyrase 1, sister telomere resolution is blocked. Unexpectedly, cohered sister telomeres become deprotected and are inappropriately fused. In contrast to telomeres rendered dysfunctional by TRF2, which engage in chromatid fusions predominantly between chromatids from different chromosomes (Bailey, S.M., M.N. Cornforth, A. Kurimasa, D.J. Chen, and E.H. Goodwin. 2001. Science. 293:2462–2465; Smogorzewska, A., J. Karlseder, H. Holtgreve-Grez, A. Jauch, and T. de Lange. 2002. Curr. Biol. 12:1635–1644), telomeres rendered dysfunctional by tankyrase 1 engage in chromatid fusions almost exclusively between sister chromatids. We show that cohered sister telomeres are fused by DNA ligase IV–mediated nonhomologous end joining. These results demonstrate that the timely removal of sister telomere cohesion is essential for the formation of a protective structure at chromosome ends after DNA replication in S/G2 phase of the cell cycle

Investigating the role of the Est3 protein in yeast telomere replication

The Est3 subunit of yeast telomerase, which adopts a predicted OB-fold, is essential for telomere replication. To assess the possible contributions that Est3 might make to enzyme catalysis, we compared telomerase activity from wild type and est3-Δ strains of Saccharomyces castellii, which revealed that loss of the Est3 subunit results in a 2- to 3-fold decline in nucleotide addition. This effect was not primer-specific, based on assessment of a panel of primers that spanned the template of the S. castellii telomerase RNA. Furthermore, using nuclear magnetic resonance chemical shift perturbation, no chemical shift change was observed at any site in the protein upon addition of single-stranded DNA, arguing against a role for Est3 in recognition of telomeric substrates by telomerase. Addition of exogenous Est3 protein, including mutant Est3 proteins that are severely impaired for telomere replication in vivo, fully restored activity in est3-Δ telomerase reactions. Thus, Est3 performs an in vivo regulatory function in telomere replication, which is distinct from any potential contribution that Est3 might make to telomerase activity

Mouse SLX4 Is a Tumor Suppressor that Stimulates the Activity of the Nuclease XPF-ERCC1 in DNA Crosslink Repair

SLX4 binds to three nucleases (XPF-ERCC1, MUS81-EME1, and SLX1), and its deficiency leads to genomic instability, sensitivity to DNA crosslinking agents, and Fanconi anemia. However, it is not understood how SLX4 and its associated nucleases act in DNA crosslink repair. Here, we uncover consequences of mouse Slx4 deficiency and reveal its function in DNA crosslink repair. Slx4-deficient mice develop epithelial cancers and have a contracted hematopoietic stem cell pool. The N-terminal domain of SLX4 (mini-SLX4) that only binds to XPF-ERCC1 is sufficient to confer resistance to DNA crosslinking agents. Recombinant mini-SLX4 enhances XPF-ERCC1 nuclease activity up to 100-fold, directing specificity toward DNA forks. Mini-SLX4-XPF-ERCC1 also vigorously stimulates dual incisions around a DNA crosslink embedded in a synthetic replication fork, an essential step in the repair of this lesion. These observations define vertebrate SLX4 as a tumor suppressor, which activates XPF-ERCC1 nuclease specificity in DNA crosslink repairope

Loss of p16Ink4a Function Rescues Cellular Senescence Induced by Telomere Dysfunction

p16Ink4a is a tumor suppressor and a marker for cellular senescence. Previous studies have shown that p16Ink4a plays an important role in the response to DNA damage signals caused by telomere dysfunction. In this study, we crossed Wrn−/− and p16Ink4a−/− mice to knock out the p16Ink4a function in a Wrn null background. Growth curves showed that loss of p16Ink4a could rescue the growth barriers that are observed in Wrn−/− mouse embryonic fibroblasts (MEFs). By challenging the MEFs with the global genotoxin doxorubicin, we showed that loss of p16Ink4a did not dramatically affect the global DNA damage response of Wrn−/− MEFs induced by doxorubicin. However, in response to telomere dysfunction initiated by the telomere damaging protein TRF2ΔBΔM, loss of p16Ink4a could partially overcome the DNA damage response by disabling p16Ink4a up-regulation and reducing the accumulation of γ-H2AX that is observed in Wrn−/− MEFs. Furthermore, in response to TRF2ΔBΔM overexpression, Wrn−/− MEFs senesced within several passages. In contrast, p16Ink4a−/− and p16Ink4a−/−Wrn−/− MEFs could continuously grow and lose expression of the exogenous TRF2ΔBΔM in their late passages. In summary, our data suggest that in the context of telomere dysfunction, loss of p16Ink4a function could prevent cells from senescence. These results shed light on the anti-aging strategy through regulation of p16Ink4a expression

Telomere disruption results in non-random formation of de novo dicentric chromosomes involving acrocentric human chromosomes

Copyright: © 2010 Stimpson et al.Genome rearrangement often produces chromosomes with two centromeres (dicentrics) that are inherently unstable because of bridge formation and breakage during cell division. However, mammalian dicentrics, and particularly those in humans, can be quite stable, usually because one centromere is functionally silenced. Molecular mechanisms of centromere inactivation are poorly understood since there are few systems to experimentally create dicentric human chromosomes. Here, we describe a human cell culture model that enriches for de novo dicentrics. We demonstrate that transient disruption of human telomere structure non-randomly produces dicentric fusions involving acrocentric chromosomes. The induced dicentrics vary in structure near fusion breakpoints and like naturally-occurring dicentrics, exhibit various inter-centromeric distances. Many functional dicentrics persist for months after formation. Even those with distantly spaced centromeres remain functionally dicentric for 20 cell generations. Other dicentrics within the population reflect centromere inactivation. In some cases, centromere inactivation occurs by an apparently epigenetic mechanism. In other dicentrics, the size of the alpha-satellite DNA array associated with CENP-A is reduced compared to the same array before dicentric formation. Extrachromosomal fragments that contained CENP-A often appear in the same cells as dicentrics. Some of these fragments are derived from the same alpha-satellite DNA array as inactivated centromeres. Our results indicate that dicentric human chromosomes undergo alternative fates after formation. Many retain two active centromeres and are stable through multiple cell divisions. Others undergo centromere inactivation. This event occurs within a broad temporal window and can involve deletion of chromatin that marks the locus as a site for CENP-A maintenance/replenishment.This work was supported by the Tumorzentrum Heidelberg/Mannheim grant (D.10026941)and by March of Dimes Research Foundation grant #1-FY06-377 and NIH R01 GM069514