The terminal end or "telomere" of every linear eukaryotic chromosome is a focal point for controlling the cell's lifespan and capacity for proliferation. There are severe consequences for human cells when telomere maintenance is disrupted, including unregulated chromosome degradation, end-to-end fusions, and aberrant recombination that pave the way for oncogenic transformation. In -90% of human cancers, the increased expression of telomerase further facilitates tumorigenesis by providing a selective advantage over surrounding cells that are normally limited by the length of their telomeres. Protection of telomeres and regulation of telomerase relies on a specialized, but still poorly understood, chromatin structure at the chromosome terminus. To date, a focus on individual genes has dominated the landscape of studies examining the link between telomeres and genome instability. However, a unified picture that pieces these separate components together into a genome-stabilizing structure has yet to emerge. This proposal outlines three approaches to determining the composition of telomere chromatin and tracking assembly of its components at chromosome ends in vivo using budding yeast. Specifically, the goals of this proposal are to (1) perform a novel genetic screen to identify proteins that physically associate with telomeres in yeast, (2) determine the composition of affinity-purified telomeric chromatin, and (3) develop an in vivo labeling methodology to monitor assembly of protein complexes at the telomere. The ultimate goal of these aims is to integrate a set of complementary, large-scale approaches to studying telomeric chromatin to understand its role in genome maintenance. Since many proteins with roles at the telomere are conserved, tracking their assembly into the unique chromatin structure at chromosome ends in yeast should contribute to understanding telomere function in human cells as well. Relevance to Public Health: The assembly of specific proteins at chromosome ends, called telomeres, plays a key role in controlling the growth of cells and their ability to divide. Alternations in telomere function contribute to cancer, as well as several genetically inherited diseases. Therefore, studies aimed at understanding the structure and function of protein complexes at chromosome ends will contribute to the development of effective preventative and therapeutic approaches to several aspects of human health. Following my doctoral research, which combined structural biology and biochemistry to understand the biophysical details of protein function, I sought to balance my skills with cellular approaches that will allow me attack problems in genome maintenance with expertise that ranges from the high-resolution structural level to the in vivo functions of large, multi-protein complexes. The goal of my postdoctoral training is to learn the latest genetic and molecular biology techniques for studying telomere chromatin in order to understand its role in stabilizing chromosomes and controlling cell proliferation. My proposal encompasses three aims each employing technically different approaches to accomplish this goal, from genetic screening to monitoring protein interactions in vivo. These aims provide excellent opportunities to broaden both my technical and scientific skills. First, I will learn the robust genetic and molecular biology tools available in S. cerevisiae, which will build upon my foundation of biophysical and in vitro techniques acquired during my predoctoral work. Second, they will expand on my background in bacterial DNA metabolism to address fundamental biological questions in a different research organism, S. cerevisiae. I have specifically chosen budding yeast for these studies because it provides an excellent experimental system to study the importance of telomeres to health-related processes, since many components of yeast 19. maintenance. In particular, discovery of the catalytic subunit of telomerase in yeast by the Lundblad lab contributed to the identification of the human telomerase reverse transcriptase. Since many proteins with roles at the telomere are conserved, the goals of my proposal to determine the protein composition and critical interactions formed at chromosome ends in yeast will be valuable to understanding telomere function in human cells. At the Salk Institute, there is an exceptional set of opportunities to expand my training outside of the laboratory. For example, weekly meetings with other groups in the Renato Dulbecco Cancer laboratories that focuses on the molecular basis of human health issues. Participating in their discussions and presenting my own work to professors, postdocs, and graduate students exposes me to other topics in cancer biology outside the central focus of my research on a regular basis. The Salk also hosts annual international symposia I will attend during my postdoctoral training that gather established scientists in the cancer biology field, such as the Mechanisms and Models of Cancer Meeting and Cell Cycle Meeting. I will also be attending conferences outside the Salk, such as the 2009 Cold Spring Harbor Laboratory Telomeres and Telomerase meeting. Finally, the Salk offers several courses open to postdoctoral trainees focused on cancer biology and therapeutics that, when combined with the opportunities mentioned above, contribute to a diverse and challenging training environment My long-term career goal is to lead an independent research lab where I will continue to pursue my commitment to understanding the role of chromosome dynamics in controlling cell lifespan and proliferation. The link between telomere integrity and cancer formation is a central hypothesis in the telomere field, but the absence of large-scale techniques for dissecting telomere structure has made it difficult to study. Each of the techniques that I propose for my postdoctoral work develops new technologies for undertaking these studies in cells. Once these new approaches are up and running, I plan to apply them in my own research program to ask questions of how telomere structure/composition changes when telomeres integrity breaks down as a model for the early events of oncogenic transformation. Experience working with S. cerevisiae will be a valuable asset for developing such models, because of its established genetic tractability and amenability to a broad range of techniques. The aims of my proposal are focused on gaining expertise in these techniques and developing a new set of tools for addressing questions in telomere biology. Together, a model for telomere breakdown and large-scale approaches for studying chromatin dynamics at telomeres will make a strong contribution to understanding genome instability in cancer formation.