Identification of novel mammalian factors involved in telomere-induced chromosome instability and telomere-damage signaling
01 / 2007 - 08 / 2012
Telomeres control replicative lifespan and protect chromosome ends from being recognized as damaged DNA. Dysfunctional telomeres activate a potent DNA-damage response that inhibits tumorigenesis but are also subject to DNA-repair activities that can result in extensive genome instability and promote tumorigenesis. The purpose of this project is to increase our understanding of how mammalian cells precisely perceive and respond to telomere-damage. To identify novel mammalian factors involved in telomere-induced chromosome instability and telomere-damage signaling we will undertake both a candidate approach and an unbiased, recently developed screening approach using RNA-interference to silence gene expression. In these different loss-of-function genetic approaches we use inhibition of the telomere protein TRF2 as a well defined and controllable experimental model system for telomere-dysfunction. In an automated high-througput single-well format we will assay a preselected set of siRNAs, inhibiting genes with a potential role at human telomeres, for their ability to prevent the accumulation of DNA-damage response factors at uncapped telomeres. In addition to this candidate-driven approach we will perform two different ?barcode? shRNA screens which have the power to identify unsuspected activities that act on unprotected telomeres. In these screens we will identify novel factors that contribute to telomere-induced genome instability and novel factors involved in the telomere-damage response by selecting shRNA vectors that attenuate the cell death or growth-arrest response to TRF2-inactivation. Factors that act directly at the telomere and/or upstream in the telomere-damage response are validated in a replicative senescence model and mechanistically investigated on the molecular, cell biological and biochemical level. These studies will significantly increase our understanding of the cellular and chromosomal consequences of telomere-dysfunction. As telomere-dysfunction is involved in aging and cancer, we anticipate to reveal novel targets for anticancer treatment strategies and novel insights in the causes and possible treatment of age-related diseases and premature-aging syndromes.