Project 5: Cellular genetic discovery for necessary and sufficient clock components

Dr. John Hogenesch and Dr. Joseph Takahashi, Project Leaders

Current efforts at understanding circadian clock mechanism are hampered by the lack of a quantitative assessment of the action of known components on oscillator function as well as an incomplete repertoire of components. Here we aim to address these limitations by first assessing gene dosage effects of known clock components using RNAi and over-expression (cDNA) on the functioning of the oscillator in peripheral mammalian cells. Oscillator function will be assayed using end-point transcriptional readouts, as well as quantitative kinetic studies aimed at assaying important parameters of clock function such as period length and amplitude. Secondly, after characterizing known components, we will attempt to discern the important parameters linking the primary output loop (Clock/Bmal1 E-box driven), and a recently described secondary loop of the oscillator (Rev-erb-alpha, Rora, RORE driven). Finally, we will endeavor to perform genome-wide screens in these cells for additional components of the circadian clock. Putative new components will be validated in animal models for their roles in central and peripheral clock function. Novel targets derived from experimental and modeling studies will be prioritized for small molecule screening and will be added as candidates for human sleep patient sequence polymorphism analysis.