PROJECT SUMMARY/ABSTRACT Master regulator transcription factors promote genetic programs required for developmental processes, enabling precise control of cell fate. Because of their importance, their misexpression in the wrong cellular context can lead to disease. One example of a transcription factor involved in such complex regulation is Double Homeobox Protein 4 (DUX4), which is crucial for early embryonic development. DUX4 is briefly expressed at the 4-cell stage in the human embryo where it activates early zygotic gene expression; whereas mis-expression of DUX4 in skeletal muscle is toxic, causing facioscapulohumeral muscular dystrophy (FSHD). We also recently identified DUX4 re-activation in multiple solid cancers. Ectopic DUX4 expression in both FSHD and cancer occurs at low levels and in very few cells but is sufficient to drive an embryonic gene program. As part of this gene network, we find that DUX4 activates histone demethylase KDM4E and histone variants H3.X and H3.Y, resulting in changes to the chromatin landscape. Furthermore, a brief pulse of DUX4 triggers long-term protein suppression, including that of Major Histocompatibility Complex class I (MHC-I) immune response proteins that may contribute to cancer immune evasion. I hypothesize that the transient expression of DUX4 initiates a perdurant naïve developmental state by establishing a chromatin memory and activating long-lived intermediary regulatory factors, and that the perdurance of the DUX4 network is a major driver of disease pathogenicity. I propose to identify epigenetic and post-transcriptional mechanisms by which DUX4 promotes its developmental program, resulting in cellular dysfunction in models of FSHD and cancer. Given the low levels and sporadic nature of endogenous DUX4 expression in FSHD and cancer cells, downstream mechanisms are difficult to elucidate in disease cell lines. My approach uses focused experiments in cellular models of DUX4 expression to elucidate epigenetic dynamics and post-transcriptional mechanisms activated by DUX4. I will: 1) use genome- wide approaches to identify epigenetic mechanisms induced by DUX4 that alter the chromatin landscape and regulate gene expression; 2) perform a targeted proteomics screen to determine the functional relevance of DUX4-target genes involved in the ubiquitin-proteasome pathway; and 3) carry out a large-scale CRISPR/Cas9 screen to identify genes responsible for MHC-I suppression. My goal is to understand the downstream biological consequences of DUX4 expression in disease and to identify mechanisms induced by DUX4 that contribute to perdurance of its early developmental program. This program consists of common molecular mechanisms employed in both early embryo development and tumorigenesis—including epigenetic reprogramming, global changes in gene expression, and immune suppression. Through a better understanding of the role DUX4 plays in development and disease, we may reveal novel diagnostic and therapeutic t...