Date of Award
Circadian clocks are oscillator systems that drive rhythms in behavior and physiology in an organism to coordinate with the light-dark cycle. The molecular clock is made up of core clock genes acting in a transcriptional negative feedback loop. Transcriptional activators BMAL1 and CLOCK form a heterodimer which binds to cis E/E-box elements present in the promoter region of target genes including Per1,2,3 and Cry1,2. PER and CRY proteins then translocate to the nucleus to repress BMAL1/CLOCK-mediated transcription. This transcriptional negative feedback mechanism underlies circadian oscillations. BMAL1 and CLOCK are bHLH and PAS domain transcription factors and play a central role in the positive limb of the negative feedback loop. In cell-autonomous clock models, absence of Clock leads to loss in rhythmicity. Previous studies and our recent results have demonstrated that Npas2, a Clock paralog, cannot complement Clock function in tissue- or cell-autonomous clock models including peripheral tissues, cells and dissociated SCN neurons. Promoted by our previous studies on BMAL1 biochemistry, we systematically investigated the CLOCK protein. We use cell-based genetic complementation and real-time bioluminescence recording and demonstrate that Clock alone can enable and maintain cell-autonomous circadian rhythms, while Npas2 cannot. Despite similarities in sequence, domain structure and biochemical activity, they play distinct roles in the clock function. We demonstrate that the C-terminal regions of CLOCK, downstream from the core bHLH and PAS domains, therefore named here the C-terminal Regulatory Domain (CRD), is key in enabling circadian oscillation and rendering its core clock function. We further identify a ~60 amino acid residue region, encompassing two motifs, within the CLOCK CRD that are required for clock function and distinguish CLOCK from NPAS2. These findings provide novel insights into the evolution of the diverse functions of the bHLH/PAS family of proteins involved in the circadian cycle and offer new opportunities for mechanistic studies of CLOCK function.
dissertation or thesis originally submitted to the local University of Memphis Electronic Theses & dissertation (ETD) Repository.
Kathale, Nimish, "BIOCHEMICAL CHARACTERISATION OF THE OSCILLATORY FUNCTION OF CLOCK" (2018). Electronic Theses and Dissertations. 1889.