PROJECT SUMMARY The traditional view of cellular metabolism is that metabolic reactions exist to maximize catabolic (energy generation) or anabolic (growth) states. Over the past decade, we and others have begun to appreciate that metabolism also regulates chromatin function, as chemicals that modify DNA and histones are derived from intermediates of cellular metabolism. Furthermore, certain metabolites non-canonically function as substrates, co-factors, and/or inhibitors of enzymes that modify chromatin. Collectively, this prior work supports a framework in which fluctuations in metabolites influence the deposition and removal of chromatin modifications. Indeed, numerous recent studies have shown that metabolites can influence cell fate via effects on chromatin organization. However, given the broad array of cellular metabolites and recent evidence that some metabolic enzymes can localize to the nucleus, this field is in its infancy. My research seeks to understand how changes in intra- and extracellular metabolites affect chromatin biology. For this MIRA R35, we will focus on polyamines, positively charged metabolites present at high concentrations in eukaryotic cells but whose effect on chromatin has been largely unexplored. Substantial work has shown that polyamines are critical for cell growth, survival, and proliferation and that polyamines interact with negatively charged macromolecules such as DNA and RNA. Moreover, interactions between polyamines and DNA can alter chromatin accessibility and increase transcriptional efficiency in vitro, but whether and how this occurs in vivo is unknown. Inhibition of polyamine biosynthesis has been known to lead to terminal differentiation of untransformed progenitors. However, the role of polyamines in cell fate and their interactions with chromatin remain unknown, largely due to prioritization of transcription factors as regulators of cell fate and the lack of tools to study metabolism at a cellular or subcellular level. Using new methods developed by our group, we recently discovered that polyamines concentrate in the nucleus, where they localize to chromatin subdomains, suggesting their interaction with specific chromatin regions. These findings lead us to hypothesize that polyamines increase chromatin accessibility at nuclear subdomains, thus impacting cell fate and chromatin homeostasis. Within this framework, we propose that polyamines act in concert with transcription factors to influence large-scale chromatin dynamics during cell fate decisions. We will explore how polyamines impact cell differentiation and reprogramming, DNA replication dynamics, replicative stress, nuclear structure, and chromatin landscape. Results from this study will help explain how polyamines regulate chromatin function, in alignment with multiple NIGMS priority areas, to better understand chromatin modification and epigenetic mechanisms. Support from this MIRA R35 will also further my independent research program in inv...