Project Summary Genome sequencing has implicated novel candidate tumor suppressors, including DAXX and ATRX which are mutated in 43% of pancreatic neuroendocrine tumors (PanNETs). Despite this strong disease association, our understanding of the physiological functions of DAXX and ATRX remains limited. Biochemical and cell biological studies have demonstrated that together they regulate the epigenome as a chaperone complex for the histone variant H3.3, depositing H3.3 in heterochromatin. Additional non-histone chaperone functions have been reported for both genes. We recently developed a conditional mouse model for Daxx and demonstrated that Daxx loss creates a permissive state that cooperates with environmental stress (inflammation) and other genetic lesions (Men1 loss) to affect the transcriptional profile and impact cell state, which ultimately impairs pancreas recovery and regeneration in vivo. The transcriptional changes are associated with dysregulation of ERVs, thereby expanding our understanding of the somatic regulation beyond DNA and histone methylation in vivo, and implicating ERV dysregulation of pancreatic neuroendocrine tumorigenesis. Importantly, we also demonstrated that dysregulation of endogenous genes near ERVs is also observed in human PanNETs with DAXX mutations. This proposal expands on these studies to better understand how mutations in DAXX contribute to tumorigenesis. We will first use our robust physiological mouse models to obtain a comprehensive understanding of the epigenetic mechanisms that underlie ERV dysregulation downstream of Daxx loss. We will conduct an integrative analysis of H3.3 incorporation, DNA methylation, and histone methylation. We will complement these genomic studies with genetic experiments to directly interrogate the Daxx:H3.3 and Daxx:Atrx interactions in vivo using newly generated mouse alleles. Finally, we will extend our studies to an innovative human pluripotent stem cell model that can be differentiated to an endoderm lineage and subsequent pancreas and endocrine cell states. This will reveal context-dependent effects of DAXX loss and illuminate the specific downstream changes that contribute to tumorigenesis. The K22 Career Transition award will not only advance these research objectives, but it will also help achieve my long-term career goal of directing a productive and impactful independent research program focused on the molecular mechanisms that underlie tumorigenesis. Specifically, it will support the establishment of my independent laboratory at a new sponsoring institution. It will further allow for the submission of my first senior author manuscript and provide the foundations and preliminary data to rapidly apply and be competitive for R01 funding. In summary, this proposal supports both research and career development in the epigenetic regulation of cellular homeostasis and how dysregulation contributes to pancreatic neuroendocrine tumorigenesis.