Project Summary/Abstract The overall goal of my research program is to mechanistically understand how circadian and metabolic rhythms shape how fully differentiated cells become functionally specialized, or “mature”. Cycles in energy availability, due to earth’s 24-hour rotation, are the oldest, most consistent environmental input for life on earth. Critical cellular functions—from gene expression to protein synthesis—are thus optimized to 24-hour rhythms as a key adaptation to daily life, from bacteria to most cells in our body. How such rhythmic physiology determines maturity is central to cell and developmental biology, and can be harnessed to build fully functional tissues for regenerative medicine. In the past decade, chronobiology studies have not only revealed novel insights into mammalian cell biology, but also identified several uncharted areas with abundant opportunities for further investigation. For the next five years, we will focus on two such areas: 1) How do circadian and metabolic rhythms determine cell maturation? 2) How do circadian and metabolic rhythms maintain cell maturity? We will address these questions in defined human in vitro and mouse in vivo model systems through the following projects: a) using in situ multimodal mapping of single-cell gene expression and functional states, we will determine how several circadian clock transcription factors program, synchronize, and entrain maturing cellular activities; b) using in vivo multiplexed gene editing, we will elucidate the molecular mechanisms by which clock components and feeding sustain mature tissue chronophysiology. Since chronic misalignment between endogenous and external rhythms triggers multi-system dysfunction (e.g., metabolic, cardiovascular, and neural syndromes), we believe that cracking the mechanisms of circadian and metabolic rhythms in the acquisition and maintenance of mature cell phenotypes will not only reveal critical knowledge to chronobiology, but may also result in act