Insight into the Regulation of Telomerase Access to Telomeres by Shelterin Proteins
Recruitment to telomeres is a pivotal step in the function and regulation of human telomerase. Impaired telomerase function can lead to premature organismal aging, development of cancer and multisystem disorders such as dyskeratosis congenita. Telomerase access to telomeres may be regulated by a telomere-binding complex termed shelterin, which is composed of TRF1, TRF2, RAP1, TIN2, TPP1 and POT1 proteins. However the molecular basis for human telomerase recruitment to telomeres is not known. Here, we have directly investigated the process of telomerase recruitment via chromatin immunoprecipitation (ChIP) and fluorescence in situ hybridization (FISH). We find that depletion of two components of the shelterin complex – TPP1 and the protein that tethers TPP1 to the complex, TIN2 – results in a loss of telomerase recruitment. On the other hand, we find that the majority of the observed telomerase association with telomeres does not require POT1, the shelterin protein that links TPP1 to the single-stranded region of the telomere. Furthermore, we find that the double-stranded telomere binding protein, TRF2 is dispensable for telomerase association with telomeres. Deletion of the oligonucleotide/oligosaccharide-binding fold (OB-fold) of TPP1 further disrupts telomerase recruitment. In addition, while loss of TPP1 results in the appearance of DNA damage factors at telomeres, the DNA damage response per se does not account for the telomerase recruitment defect observed in the absence of TPP1. Our findings indicate that TIN2-anchored TPP1 plays a major role in the recruitment of telomerase to telomeres in human cells and that the recruitment does not depend on POT1 or interaction of the shelterin complex with the single-stranded region of the telomere. We propose that the loss of TRF2 can be compensated by the second double-stranded telomere binding protein, TRF1 that anchors TIN2/TPP1-recruited telomerase onto the telomeres. Dyskeratosis congenita (DC) is a multisystem disorder characterized with bone marrow failure, cancer predisposition and defective telomere maintenance. The TINF2 gene that encodes for the TIN2 shelterin protein is one of seven mutated genes identified in DC patients. Here, we have examined the effects of TIN2 DC mutants on telomere protection and shelterin protein stability. We find that exogenous expression of TIN2 DC mutants can rescue the TIN2-depleted phenotype, restoring TPP1 protein levels and telomere protection. Our findings indicate that TIN2 DC mutants preserve the intact telomere structure and protection. We propose that defective telomere elongation may be the underlying cause of impaired telomere maintenance in TIN2 DC patients.
Keywords: telomerase ; telomeres ; shelterin ; TPP1 ; telomerase recruitment ; TIN2 ; dyskeratosis congenita ; télomérase ; télomères ; 'shelterin' ; TPP1 ; recrutement de la télomérase ; TIN2 ; dyskératose congénitaleThèse École polytechnique fédérale de Lausanne EPFL, n° 4982 (2011)
Programme doctoral Approches moléculaires du vivant
Faculté des sciences de la vie
Institut suisse de Recherche Expérimentale sur le Cancer
Unité du Prof. Lingner
Record created on 2011-01-06, modified on 2016-08-09