Project Summary/Abstract Telomere length plays a pivotal role in cancer and age-related degenerative disease. Telomeres are made of simple tandem repeats, added by the enzyme telomerase, to establish a length distribution. A mechanistic understanding of the forces that regulate telomere length is needed to develop approaches to disease. Current models for how the telomere length equilibrium is maintained suggest that ends with fewer telomere repeats are more frequently elongated than ends with many repeats implying that all telomeres are regulated around the same length distribution. We developed a nanopore sequencing method to measure telomere length in yeast at the nucleotide level. Surprisingly, we found that each telomere had a distinct length distribution that was maintained over hundreds of cell divisions. This discovery requires that current models for length regulation be revised. In this proposal we detail specific experiments to test mechanisms that may modify the length distribution at specific chromosome ends: 1. Subtelomeric sequences 2. Telomere transcription/telomere RNA (TERRA), and the Sir2/3/4 histone modification complex. We will use computational approaches to determine if specific sequence motifs are associated with long or short telomeres, and we will systematically delete DNA binding sites to test their role. We will use nanopore RNA sequencing to study the long non-coding RNA TERRA, and test whether the RNA, or the action of transcribing the telomere play a role in end-specific telomere lengths. Finally, we will test the role of the histone deacetylase Sir2 and the interaction of Sir4 with Ku in determining end-specific telomere lengths. These experiments will implicate or rule out the specific factors that establish different length distributions. Understanding molecular mechanism(s) that influence telomere length distributions will set the stage to specifically manipulate telomere length. Because mechanisms of telomere length regulation are conserved across species, our experiments will set the stage for approaches to alter telomere length to treat human disease.