Project Summary Human telomeres are specialized chromatin structures uniquely programmed to coordinate cellular division with genomic integrity, thus providing a senescence barrier to cancer-cell evolution. Telomere structural maintenance and genome sensing functions require extensive epigenetic signaling and higher-order chromatin dynamics that are poorly understood. Our previous studies have revealed the important roles of telomere encoded repeat RNA (TERRA) in the coordinate regulation of telomere chromatin structure, DNA replication and repeat-length maintenance. We have found that TERRA expression can be regulated by subtelomeric binding of chromatin organizing factor CTCF and tumor suppressor protein p53 in response to DNA damage stress. TERRA is also regulated by histone assembly factors DAXX and ATRX that are implicated in the Alternative Lengthening of Telomeres (ALT) through recombination. New preliminary data indicates that DAXX and ATRX are also required for p53 binding to subtelomeres in response to DNA damage, suggesting a coordinate regulation of these factors in telomere chromatin control and TERRA expression. In Aim 1, we investigate the role of DAXX and ATRX in regulating telomeric and subtelomeric chromatin accessibility that is critical for p53 binding, TERRA expression, and telomere DNA repeat stability. TERRA is also known to interact with shelterin component TRF2. In Aim 2, we propose to investigate the interaction of TERRA with the TRF2 amino terminal GAR domain, and how this further regulates telomeric DNA replication, chromatin structure, and chromosome conformation. Finally, in Aim 3, we will investigate how TERRA and telomeric chromatin can be processed into vesicular bodies in response to telomeric stress to generate extracellular inflammatory signals. We propose that exosomal TERRA and telomeric chromatin serve as telomere-specific Alarmins that are important in cellular senescence and tumor microenvironment signaling. These studies will provide an integrated analysis of telomere transcription and chromatin regulation, with stress response pathways that are important in human cancer and related inflammatory disease. Central Hypothesis: Telomeres are specialized epigenetic elements that sense and signal replication stress to control cell proliferative capacity and senescence. TERRA represents an important telomere-repeat generated molecule that functions in chromatin structure, DNA replication and repair, and DNA damage signaling. Characterization of normal and pathogenic functions of TERRA will provide important insight into the mechanisms driving genetic instability in cancer and related genetic instability syndromes.