The broad, long term objective of this research is to understand the molecular mechanisms by which telomeres function. Telomeres, the ends of eukaryotic chromosomes, are important for chromosome integrity. The regulation of telomere length and telomerase has been implicated in the treatment of cancer cells and in the manipulation of cell life span. An important aspect of telomere function is telomeric silencing, the repression of genes that are located adjacent to telomeres. Since some of the factors involved in yeast telomeric silencing are highly conserved or have structural homologs in humans, understanding the mechanisms by which silent chromatin becomes, and remains, epigenetically silenced is likely to reveal new paradigms that underlie the mechanisms of developmentally regulated gene silencing and parental imprinting in humans. A major goal for the next grant period is to elucidate the molecular mechanisms for the establishment, maintenance and inheritance of telomeric silencing. In the past grant period, a novel phenotype that provides an extraordinarily sensitive measure of the heterochromatin state at HML (a silent mating locus) was identified. In the next grant period we will develop and use a new assay that brings the single cell resolution and accompanying insights that we have gained at HML to the study of silencing at telomeres. In the previous grant period, genes identified in the TEL+CEN-antagonism screen revealed previously unknown connections between telomere functions and two processes: chromatin assembly and nonsense-mediated mRNA decay. These studies suggest a model for silencing in which the timing of chromatin assembly, as well as the geometry and stoichiometry of the silent chromatin components, affect the formation of silent chromatin. We will test this model, with special emphasis on the genes identified in our screens. In the next grant period we will continue to study these genes, focusing on the mechanisms by which they influence telomeric silencing and telomere length control. Specific Aims 1, 2 and 3 are designed to study mechanistic issues of silencing. In Aim 1, we will compare telomeric and HML silencing at the single cell level. In Aim 2, we will study temporal issues of silencing, especially as they relate to chromatin assembly. In Aim 3, we will explore the inheritance of silencing by asking how histone mutants influence silencing. Aim 4 addresses a different aspect of the regulation of telomere structure and function by asking how telomeres are regulated by the nonsense mediated mRNA decay pathway and whether this connection between the nonsense mediated mRNA decay pathway and telomeres is conserved through evolution.