Circadian clocks influence nearly all aspects of mammalian life, aligning our internal physiological process to optimal times of day. Understanding the molecular circuitry keeping circadian time provides insight into how the clock drives overt rhythms, and what to fix when the circadian system is disrupted (i.e. during shift work). Circadian time coded in the rhythmic regulation of clock gene expression in a negative feedback loop system. Critical to this timing system is the circadian degradation of rhythmically abundant clock proteins; however these mechanisms have remained elusive. To begin elucidating these mechanisms, we developed a new functional screening approach to identify which E3 ubiquitin ligases degrade which clock proteins and screened for E3 ligases that degrade RevErb?/? proteins. Both RevErb proteins are essential for normal clock function and exhibit high amplitude abundance rhythms, but the mechanisms driving their circadian clearance are unknown. Our screen identified two novel candidate RevErb E3 ligases, Spsb4 and Siah2, and preliminary data suggest these E3s contribute directly to the rhythmic degradation of RevErb proteins. Moreover, our data suggest for the first time that the rate of circadian RevErb degradation is a determinant of circadian periodicity. Identifying the roles, mechanisms and contributions to overall clock function of Spsb4 and Siah2 is a major focus of our current research proposal. Bolstered by the fact that both hits from this screen appear to be genuine regulators of RevErb stability, the other focus of our proposal is to expand our screening efforts. Experiments proposed in Specific Aim 1 focus on elucidating the mechanisms of Siah2/Spsb4 degradation of RevErb proteins in the context of an oscillating cellular system. The experiments proposed in Specific Aim 2 delve deeper into the role of Siah2-mediated degradation of RevErb proteins in overall clock function in vivo. Experiments in Specific Aim 3 will identify candidate E3s for all of the remaining essential core clock proteins using an expanded E3 ligase cDNA screening library. Success in these aims will provide essential background and validation of our future efforts to identify protein degradation mechanisms. Overall, uncovering the novel mechanisms mediating rhythmic degradation of clock proteins will open many new avenues for treating circadian-related disorders.
Shift work and jet lag can cause a variety of diseases by disrupting our internal circadian timekeeping system. Circadian timekeeping is coded by the rhythmic expression and degradation of so-called 'clock proteins', but mechanisms regulating degradation of most clock proteins have remained elusive. The experiments proposed in our application will define the degradation for two essential clock proteins and identify regulators fo other essential clock proteins.
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