Characterization of core clock complexes in time and space using quantitative proteomics

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Circadian clocks are cell endogenous self-sustainable oscillators found in virtually every cell in the body that play a fundamental role in cellular and tissue physiology. Circadian clocks allow our organs to molecularly anticipate and prepare for recurrent needs across the day associated to our cycling environment. The molecular mechanisms driving these oscillations comprise transcriptional and translational feedback loops. In the last years, the application of MS-based quantitative proteomics to circadian biology has broadened our understanding of how dynamics of protein abundance and function contribute to the daily regulation of cellular and metabolic processes. This technology complements and extends previously established transcriptomic studies and provides a critical novel angle in our knowledge of the molecular and cellular clocks. In addition to expression proteomics, MS-based interaction proteomics in combination with affinity purification methods has greatly contributed to the identification of novel interactors with key roles in the clock molecular mechanism. Nonetheless, there are still many unknown aspects of this mechanism, for example how the binding of the transcription factor complex BMAL1/CLOCK to promoters is regulated in a temporal and tissue dependent manner. We propose here to apply state of the art MS technologies in combination with quantitative proteomics to ultimately understand how the BMAL1/CLOCK transcriptional complex can modulate transcription in a temporal manner but also in a tissue specific context. Specifically, this grant aims to study the temporal and spatial composition of BMAL1/CLOCK complexes bound to chromatin but also in the cytoplasm of cells and mouse tissues to identify and functionally characterize novel co-regulators. Many processes contributing to circadian timing such as protein stability and protein-protein interaction are regulated by posttranslational modifications (PTMs). Thus, by taking advantage of MS-based quantitative proteomics, a powerful technique not only to identify PTMs but also to characterize their temporal dynamics, this grant also aims to identify novel oscillating post-translational modifications in clock proteins isolated from cells as well as tissues and characterize their functional role. Together, the work of this grant will shed light on temporal and spatial modulation of BMAL1/CLOCK complexes, both critical aspects for circadian function.