Senior Scholar Award in Aging
M.D. , Ph.D.
The Role of T-loops in Aging of Human Cells
In collaboration with Jack Griffith (UNC) we have found that human telomeres form large duplex loops (t-loops). We propose that t-loops represent the mechanism by which telomere ends are masked from the cellular machinery that detects DNA breaks. Our working hypothesis is that telomere shortening in aging human cells results in chromosome ends that no longer form t-loops. Such t-loop deficient termini are proposed to constitute the main signal leading to cell cycle arrest, senescence, and apoptosis in aging cells.
We have previously identified a key player at mammalian telomeres, the telomeric DNA binding factor, TRF2. Loss of TRF2 from chromosome ends results in immediate deprotection of telomeres. Cells respond to this insult with the activation of a DNA damage pathway that includes the ATM kinase and p53, resulting in cell cycle arrest. Depending on the cellular context, such cells undergo apoptosis or display a phenotype similar to senescence. The unprotected telomeres lose the 3' protrusion of TTAGGG repeats typical of mammalian chromosome ends and eventually become ligated, forming dicentric and multicentric chromosomes. Thus, the removal of TRF2 from chromosome ends mimics events observed in aging human cells in which the telomeres have become critically shortened.
A likely mechanism for TRF2-mediated telomere protection has now been revealed in a study on the structure of the telomeric complex. Using electron microscopy, we have found that telomeres can exist in a specific higher order conformation (t-loops). EM analysis of psoralen crosslinked telomeric DNA demonstrated frequent large t-loops at natural chromosome ends. Molecular studies indicate that t-loops are formed through invasion of the 3' telomeric overhang into the duplex telomeric repeat array. We propose that t-loops are the main mechanism by which cells sequesters telomere ends from DNA damage checkpoints. In agreement with its protective role at telomeres, TRF2 has the ability to promote t-loop formation in vitro, suggesting that its main function in vivo is to facilitate remodeling of telomeric DNA into the t-loop configuration.
The presence of t-loops at human chromosome ends suggests a mechanism by which telomere shortening could induce cellular senescence and apoptosis. We propose that critically shortened telomeres fail to form t-loops, resulting in unfolded, exposed chromosome ends that activate a senescence or apoptotic signalling pathway, possibly involving ATM and p53. This hypothesis will be tested by determining the relationship between t-loop loss, telomere shortening, and cellular aging phenotypes. In addition, we will aim to manipulate the presence of t-loops at chromosome ends in order to test whether loss of t-loops can induce premature senescence and apoptosis. Conversely, we will test whether improved t-loop formation can extend the in vitro life-span of human cells.
Dr. de Lange.