Mechanistic Basis of Circadian Clocks in Bmal1 Knockout Mice

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PROJECT SUMMARY Circadian clocks are believed to exist at almost all levels of life and play a fundamental role in maintenance of physiological and behavioral processes in accordance with the day-night cycle. The conventional model that describes the circadian clockwork at the molecular level revolves around transcriptional/translational feedback loops (TTFLs). In these models, BMAL1 is believed to act as an indispensable component of the timekeeping system. However, we have found pervasive molecular oscillations in the transcriptome and proteome of Bmal1-/- mice. A research program will be undertaken to obtain a comprehensive mechanistic understanding of these “non-canonical” circadian rhythms in Bmal1-/- mice. The project will broadly focus on understanding transcriptional (Aim 1) and post- transcriptional (Aim 2) functioning of cells and tissues from Bmal1-/- mice. (Aim1) Our preliminary data suggest that the novel circadian rhythms we see might be underpinned by the recruitment of ETS family transcription factors into the clockwork. We will functionally test their role by knocking out key ETS proteins using CRISPR. We will also perform ChIP-seq experiments to find genomic targets driving rhythmic transcripts that we see. In addition, we will perform nuclear proteomics to elucidate novel transcription factors that might mediate rhythmic transcription. Furthermore, we will perform protein interaction analyses using immunoprecipitation mass spectrometry to determine how ETS proteins and redox proteins may physically interact. (Aim 2) We found redox oscillations in Bmal1 knockout cells, implying that these play a role in the clockwork of Bmal1-/- mice. Consequently, we will investigate novel redox oscillations in Bmal1 knockout cells using a novel redox proteomics workflow that we have developed. In addition, we will characterize the rhythmic phospho-proteome and kinome of cells, which we have found could be profoundly affected by deletion of Bmal1. Finally, we will determine whether metabolic circadian oscillations occur in Bmal1 knockouts. Gaining new molecular insights into the circadian clockwork will guide future therapeutic interventions to alleviate the disorders associated with circadian disruption, which are highly prevalent in contemporary society.