Many physiological processes and behaviors are under circadian clock control, including sleep. However, the molecular and circuit mechanisms underlying how the circadian clock regulates these behaviors remain poorly understood. We recently identified a novel molecule in Drosophila named WIDE AWAKE (WAKE) that plays a key role in mediating the circadian timing of sleep onset. In our original grant studying this molecule, we determined that WAKE is rhythmically expressed in arousal-promoting clock neurons and acts to upregulate GABAA receptors, thus cyclically suppressing the activity of these cells to promote sleep. Interestingly, growing evidence from our group and others suggests that WAKE-related molecules broadly function to spatially organize signaling complexes in a time-dependent manner. Moreover, there is a single homolog of WAKE in mammals, including humans, which is enriched in the circadian pacemaker suprachiasmatic nucleus. Thus, insights gained from studying WAKE in flies may help unravel how the circadian system regulates sleep in mammals as well. In this renewal of our previous grant, we propose to further our understanding of the mechanisms underlying the circadian modulation of sleep, by studying additional circuit and molecular mechanisms by which WAKE modulates this process. Specifically, we plan to carry out the following aims: 1) study the role of WAKE in regulating additional WAKE-expressing circadian clock neurons, and how this regulation impacts downstream arousal circuits; 2) identify and characterize additional proteins that interact with WAKE to modulate sleep; and 3) examine how glia may interact with these WAKE-expressing circadian clock circuits to regulate sleep. We will use a multidisciplinary approach, including cell biological, genetic, behavioral, and electrophysiological assays, to perform these studies. Circadian dysregulation of sleep is estimated to impact millions of people in the U.S. and has been implicated in adverse effects on health and productivity. Developing a better understanding of how the circadian clock regulates sleep could pave the way for identifying novel therapies to treat these disorders.