Project Summary/Abstract Eukaryotic cells require the tight regulation of global gene expression to maintain homeostasis and respond to environmental stimuli. DNA spools around histone proteins form this vital structure, chromatin, and provide a platform for the sophisticated tuning of gene expression through physical and chemical regulation. Unsurprisingly, the disruption of these chromatin regulatory mechanisms is particularly prevalent in cancers as a driver of disease. Completion of the proposed projects will shed light on the mechanisms of healthy chromatin regulation and its disruption in disease, providing the insight necessary to develop improved therapeutic interventions in a variety of cancers. In the F99 phase of this proposal, I study disrupted chromatin signaling by Hepatitis B Virus (HBV), a leading cause of hepatocellular carcinoma worldwide. HBV maintains chronic infections within hepatocytes by establishing an independent minichromosome, termed covalently closed circular DNA (cccDNA), that largely evades immune detection and conventional chromatin regulatory mechanisms. Further contributing to this evasion is the viral protein HBx, which has documented roles redirecting numerous chromatin effectors, including transcription factors, degradation machinery, and epigenome modifiers. So far in my thesis work, I have developed a platform to reconstitute cccDNA in vitro for biochemical and biophysical studies, determined that histone occupancy in cccDNA is required for HBx expression, and shown that HBx binds directly to nucleosomes. The remainder of my thesis work will be spent testing the biochemical effects of other known interactors on cccDNA compaction and gene expression and illuminating the cccDNA landscape in cells using locus-specific proteomic and epigenomic studies. The K00 phase shifts focus to ATP-dependent chromatin remodeling enzymes, a class of proteins shown to be mutated or overexpressed in more than 20% of cancers. In particular, I intend to study the CHD family of remodelers, which have been implicated as drivers of a variety of cancer types. I will apply my expertise in chromatin biochemistry and expand my technical repertoire to include cryo-electron microscopy as a means to study the structure and function of CHD chromatin remodelers. In parallel, I will develop skills in gene editing techniques to knockout wild-type enzyme and introduce clinically-relevant CHD mutants into cells for epigenomic analyses of remodeler dysfunction in disease. Combining these new approaches with my background in biochemistry, chemical biology, and biophysics will position me to address pressing questions in chromatin and cancer biology throughout the rest of my career as I pursue an independent, cancer-focused faculty position.