Abstract Circadian clocks throughout the body drive rhythmic expression of thousands of genes, resulting in rhythms in biochemistry, physiology and behavior. Disruption of circadian clocks through genetics or environmental perturbations such as jet lag or shift-work, can have profound negative consequences and has been linked to obesity, diabetes, cancer, cardiovascular disease and mental illness. In particular, circadian clocks exert control over nearly every major metabolic pathway, allowing optimal utilization of typically cyclic availability of nutrients. Our work is focused generally on understanding the detailed molecular mechanisms of the mammalian circadian clock machinery and the mechanisms by which these clocks control rhythmic metabolism. According to the current model, the core part of this clock mechanism is a negative feedback loop whereby the transcription factor heterodimer CLOCK/BMAL1 drives transcription of the “clock” proteins PERIOD (PER) 1, PER 2, CRYPTOCHROME (CRY) 1 and CRY 2 which interact with each other to repress the activity of CLOCK/BMAL1, and thus their own synthesis. This same transcriptional mechanism also drives rhythmic expression of many so called “clock-controlled genes” that ultimately result in the many output rhythms in tissues throughout the body. One of these clock- controlled genes is Nocturnin, a focus of my laboratory. We have shown that loss of this gene in mice causes resistance to diet-induced obesity, alters rhythms in cholesterol and triglyceride metabolism and increases resistance to inflammatory challenges such as LPS. Although the Nocturnin protein is a member of a family of RNases (the CCR4 deadenylases), we have recently demonstrated that Nocturnin’s substrate is not RNA, but rather NADP(H). Nocturnin is an NADP(H) phosphatase, converting NADP(H) to NAD(H). In this proposal we seek to use new mouse models that we have developed to understand the tissue-specific role of Nocturnin and to disentangle the roles of the two isoforms (one mitochondrial, the other primarily bound to intracellular membranes). We will also carry out metabolomic and metabolic flux experiments to determine the effects of circadian regulation of NADP(H) and NAD(H) levels. In addition, we will validate small molecule modulators of Nocturnin that we have identified in a high throughput screen and will use these molecules to perturb Nocturnin activity in cells and mice. Our work on the central mechanism of the core clock will also continue in the proposed funding period. We have solved crystal structures for the CLOCK/BMAL1 and CRY2/PER2 complexes and these data have allowed the identification of evolutionarily conserved functional domains throughout the proteins and revealed additional insights into the mechanisms by which these proteins operate and set the circadian period. Over the next five years, we will expand on this information to determine the atomic details of how this clock keeps time.