The ends of linear chromosomes, telomeres, are structures essential for chromosome stability, aging and tumor suppression, made up of the sequence TTAGGG and interacting proteins. The very end of all telomeres consists of a single stranded overhang of the G rich 3' strand that can be up to 300 bases long. This 3' end invades the double stranded portion of the telomere, forming protective loops. Telomeres are maintained by telomerase, a reverse transcriptase that copies TTAGGG repeats to the chromosome ends. Telomerase is suppressed in most human somatic cells, leading to constant telomere shortening, giving rise to the tumor suppressive effect. The two telomeric repeat binding factors TRF1 and TRF2 bind to the double stranded portion of the telomere. The proteins interact with a number of different partners, leading to the formation of independent complexes. Both complexes have been implicated in telomere length regulation, and the TRF2 complex plays a protective role. Targeted deletion of TRF1 led to early embryonic lethality with features pointing at apoptosis, giving rise to our hypothesis that TRF1 is necessary for chromosome end protection. This proposal focuses on testing this hypothesis. Additionally, we will gain understanding in the role of TRF1 in the embryonic development.
In AIM1 we will generate in vivo and in vitro tools to analyze TRF1 functions by creating a conditional deletion of the gene. The deletion will be made in the mouse and in tissue culture cells, and these systems together with blastocyst cultures will be used to analyze the effect of the TRF1 deletion on cell growth, viability, and chromosome structure.
AIM2 focuses on structure function analysis of TRF1. We will use TRF1 mutants to rescue phenotypes resulting from the TRF1 deletion. These experiments will be performed in tissue culture systems, and moved into mice for rescue experiments.
In AIM3 we plan to isolate TRF1 interacting factors that are involved in phenotypes derived from the deletion of TRF1. The TRF1 'knockout' is the first targeted deletion of a telomeric protein available. Results from this study are expected to shed light onto the integrity of the telomeric complex, and therefore contribute to the understanding of aging and cancer development.
|O'Sullivan, Roderick J; Kubicek, Stefan; Schreiber, Stuart L et al. (2010) Reduced histone biosynthesis and chromatin changes arising from a damage signal at telomeres. Nat Struct Mol Biol 17:1218-25|
|O'Sullivan, Roderick J; Karlseder, Jan (2010) Telomeres: protecting chromosomes against genome instability. Nat Rev Mol Cell Biol 11:171-81|
|Begemann, Sebastian; Galimi, Francesco; Karlseder, Jan (2009) Moderate expression of TRF2 in the hematopoietic system increases development of large cell blastic T-cell lymphomas. Aging (Albany NY) 1:122-130|
|Raices, Marcela; Maruyama, Hugo; Dillin, Andrew et al. (2005) Uncoupling of longevity and telomere length in C. elegans. PLoS Genet 1:e30|
|Verdun, Ramiro E; Crabbe, Laure; Haggblom, Candy et al. (2005) Functional human telomeres are recognized as DNA damage in G2 of the cell cycle. Mol Cell 20:551-61|
|Crabbe, Laure; Verdun, Ramiro E; Haggblom, Candy I et al. (2004) Defective telomere lagging strand synthesis in cells lacking WRN helicase activity. Science 306:1951-3|