Senior Scholar Award in Aging
Jack D. Griffith, Ph. D.
University of North Carolina - Chapel Hill

Telomere Looping and Control of Cell Aging

Our research has focused on the long range remodeling of DNA as it undergoes replication, recombination, and repair. Such topics remain one of the most difficult areas to study using standard molecular tools but can be approached by direct electron microscopic (EM) visualization. Telomeres are believed to represent a molecular clock that regulates the number of cell divisions allowed before cellular senescence. This regulation appears to depend on the length of the telomeric DNA and thus by inference, must also depend on the physical structure of the telomere. Over the past several years in collaboration with Dr. Titia de Lange at the Rockefeller University we have asked how two proteins which bind mammalian telomeric DNA, TRF1 and TRF2, organize telomere architecture. As a result of the studies we discovered that the protruding 3' single-stranded overhang of the telomere folds back and invades the preceding telomeric duplex tract to arrange the telomere into a giant duplex loop. Telomere looping may be a general paradigm since it has now been observed in several lower eukaryotes. This discovery suggested how the architecture of the telomere could protect the telomere end from nucleases in the cell and provided a structural model of how telomeres count the number of cell divisions. Our proposed research is focused on further probing these questions based on our new knowledge of telomere architecture. Ultimately such information will help describe the processes controlling the aging clock.

Presently EM provides the only definitive assay for the arrangement of telomeres into loops and there are many pressing experiments to be done relating cellular aging to the properties of t-loops. We will grow human fibroblasts in culture through multiple generations beginning with isolation from newborn foreskin and ending in senescence. The size distribution and frequency of t-loops will be monitored throughout this process, with particular emphasis on the later stages as the cell approach senescence. EM analysis of telomere looping can be done from a single mouse liver, opening the door to studies that take advantage of the many transgenic mouse lines which contain mutations in genes related to telomere maintenance and aging. T-loop preparations will be made from mice of successive breeding generations that lack functional telomerase (in collaboration with Dr. Carol Greider) and the size of the loops determined at each generation. In studies with Dr. Titia de Lange, we will determine whether t-loops persist after the functional removal of TRF2 from cells and will synchronize cells at various points through the cell cycle we will examine the cells for the presence, and size distribution of telomere loops.

The discovery of t-loops led us to propose that the mammalian telomere is organized into a highly ordered, condensed nucleoprotein particle. In this model the normal chromosomal proteins (histones) would induce the first level of DNA packing into chromatin particles termed nucleosomes, followed by further condensation by the telomere binding proteins TRF1 and TRF2 This compact particle could sequester the t-loop junction from nucleases. We will reconstitute telomeric DNA into a chromosomal structure by incubating high molecular weight human telomeric DNA with purified histone proteins, as well as TRF1 and TRF2. This will be done using a new chromatin assembly system derived from Drosophila cells. Electron microscopy will be used to visualize the structure of the reconstituted telomeres and to determine how the structure may relate to the way in which cells are able to count the number of cell divisions.

Contact Dr. Griffith.