Telomeres provide the 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 as it is folded into a loop and then further compacted by telomere specific proteins such as TRF1 and TRF2 and the related shelterin proteins.  Understanding the structure of this particle, how it changes with the age of the cell, and how it depends on the function of specific aging-related genes is a central goal of this study.

Over the years we have  collaborated with Dr. Titia de Lange at the Rockefeller University to understand how two proteins which bind mammalian telomeric DNA, TRF1 and TRF2, organize telomere architecture. From our EM examination of TRF2-DNA complexes and consideration of how recombination proteins such as recA would interact with DNA having the sequence and architecture of a mammalian telomere, this P.I. made a novel proposal. I suggested that the protruding 3′ single-stranded overhang of the telomere would fold back and invade the preceding telomeric duplex tract to form a classic D-loop. Incubations of a model telomere DNA with purified TRF2 protein revealed looped forms in vitro. This was followed by the isolation of psoralen crosslinked telomeric DNA and EM visualization which revealed mammalian telomeres arranged into giant duplex loops (termed t-loops). Telomere looping may be a general paradigm since it has now been observed in mice, chickens, man, plants and in several lower eukaryotes. This opened a new vista into telomere structure and posed critical questions whose answers will ultimately describe the processes controlling the aging clock as well as progression of cells to an oncogenic state.
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. One research goal is to develop non-EM based assays for t-loops. EM however will remain an essential primary tool for some time. We 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 cells 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 and the size of the loops determined at each generation. Werner’s syndrome is a premature aging syndrome and the affected gene codes for a DNA helicase that could interact with the t-loop junction. We will determine whether cells from these mice have normal sized telomere loops and will determine how purified Werner’s helicase interacts with model t-loops formed in vitro. In studies with Dr. Titia de Lange, we will determine whether t-loops persist after the functional removal of TRF2 from cells. Their studies based on expression of inducible knockout mice for TRF2 provide the ideal experimental system for deducing the true role of TRF2 in the cell.

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 TRF1 and TRF2. This compact particle could sequester the t-loop junction from nucleases such as the MRE11/Rad50/NBS1 complex (presumably the loop would open at S phase for replication). When cells age and the telomeric DNA becomes critically short, the telomere particle would, we suggest, be too small to provide adequate protection at the t-loop junction. We estimate that this would occur when the telomere shortens to ~2-3 kb (15 or fewer nucleosomes). This is, indeed, close to the critical length of human telomeres at senescence. The histones bind telomeric DNA, but not as strongly as normal sequence DNA and human telomeres exhibit a nuclease digestion pattern suggestive of a tighter than usual nucleosome spacing. The uniform sequence of telomeric DNA does not present energy barriers to the histone particles in their sliding along DNA and this could generate an extremely regular packaging of the telomeric DNA which would be further condensed by TRF1 and TRF2. Our goal is to 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 chromatin assembly system derived from Drosophila cells. Several modes of EM analysis will be employed including cryo EM. These studies will be complemented by nuclease and chemical probing. We believe this to be a compelling model which could explain how the cell measures telomere length and thus regulates cellular life span. Reconstitution experiments such as these are the most direct way to test this model and EM provides the only clear means of examining such large structures.

We have recently purified ample amounts of each of the telomere-associated proteins (shelterins) including hRAP1, Pot1, Tin2, TPP1 and are poised to generate combinations of these proteins bound to model telomere templates. Using a combination of EM and biochemical methods it will be possible to answer many basic questions about the role of these proteins in protecting the telomere.

A longstanding collaboration with Dr. Lubomir Tomaska at Comenius University in Brtatislava has provided the ideal venue for us to utilize the power of yeast genetics to answer basic questions of telomere function. These active studies are continuing.

Selected References:

  • Jack Griffith, Laurey Comeau, Soraya Rosenfield, Rachel Stansel, Heidi Moss, Alessandro Bianchi, and Titia de Lange. Mammalian telomeres end in a large duplex loop. Cell: 97, 503-514. 1999.
  • Lubomir Tomaska, Alexander M. Makhov, Jozef Nosek, Blanka Kucejova, and Jack D. Griffith. Electron microscopic analysis supports a dual role for the mitochondrial telomere-binding protein of Candida parapsilosis. J Mol. Biol., 305: 61-69 2001.
  • Anthony J. Cesare, Nancy Quinney, Smaranda Willcox, Deepa Subramanian and Jack D. Griffith. Telomere Looping in P. savitum (common garden pea). The Plant Journal. 36: 271-279. 2003.
  • Lubomir Tomaska, Smaranda Willcox, Judita Slezakova, Jozef Nosek, and Jack D. Griffith. Taz1 binding to a fission yeast model telomere: formation of t-loops and higher order structures. J Biol Chem, 279: 50764-50772, 2004.
  • Anthony J. Cesare and Jack D. Griffith. Telomeric DNA in ALT cells is characterized by free telomeric circles and heterogeneous t-loops. Mol. Cell. Biol 24: 9948-9957. 2004. Cindy Groff-Vindman, Anthony J. Cesare, Shobhana Natarajan, Jack D. Griffith and Michael J. McEachern Recombination at long mutant telomeres produces tiny ss and ds telomeric circles. Mol. Cell. Biol. 11: 4406-4412, 2005
  • Nicole Fouche, Sezgin Ozgur, Desmandia Roy and Jack D. Griffith. Replication fork regression in repetitive DNAs. Nucl. Acids. Res. 34: 6044-6050, 2006
  • Nicole Fouche, Anthony J. Cesare, Smaranda Willcox, Sezgin Ozgur, Sarah A. Compton and Jack D. Griffith, The Basic Domain of TRF2 Directs Binding to DNA Junctions Irrespective of the Presence of TTAGGG Repeats. J. Biol. Chem. 281: 37486-37495, 2006
  • Sarah A Compton, Jun-Hyuk Choi, Anthony Cesare, Sezgin Ozgur, Jack Griffith. Xrcc3 and Nbs1 are required for Telomere Length Maintenance and Production of Telomeric Circles in Human ALT cells. Cancer Res, Feb 15 2007; 67(4)
  • Tomaska L, Nosek J, Kramara J, Griffith JD. Telomeric circles: universal players in telomere maintenance? Nat Struct Mol Biol. 2009 Oct;16(10):1010-5. PMID: 19809492
  • Randall A, Griffith JD. J Structure of long telomeric RNA transcripts: the G-rich RNA forms a compact repeating structure containing G-quartets. Biol Chem. 2009 May 22;284(21):13980-6.  PMID: 19329435
  • Kramara J, Willcox S, Gunisova S, Kinsky S, Nosek J, Griffith JD, Tomaska L.  Tay1 protein, a novel telomere binding factor from Yarrowia lipolytica. J Biol Chem. 2010 285(49):38078-92. PMID: 20923774
  • Compton SA, Ozgür S, Griffith JD.  Ring-shaped Rad51 paralog protein complexes bind Holliday junctions and replication forks as visualized by electron microscopy. J Biol Chem. 2010 285(18):13349-56..PMID: 20207730
  • Basenko EY, Cesare AJ, Iyer S, Griffith JD, McEachern MJ. Telomeric circles are abundant in the stn1-M1 mutant that maintains its telomeres through recombination. Nucleic Acids Res. 2010 38(1):182-9. PMID: