Wednesday, June 4, 2025
6/4/2025
BMAL1 Circadian Clock Complexes from Mammalian Brain - Project Summary The circadian clock is an endogenous ~24-hour oscillator that drives daily rhythms of biological processes. In mammals, circadian clocks are found in the brain and peripheral tissues. Together, the distributed clocks constitute the basic timing system regulating behavior, physiology, and metabolism. The mammalian circadian clock is built on a transcriptional negative feedback loop that generates circadian rhythms at the molecular level. The transcription factor BMAL1 is at the heart of this feedback loop, acting as the positive element activating circadian clock transcription. Long-standing evidence suggests that peripheral clocks and brain clocks differ with respect to BMAL1. In the periphery, BMAL1 transcriptional action (and clock function) requires the heterodimeric partner CLOCK, whereas in the brain either CLOCK or NPAS2, a protein related to CLOCK, suffices to sustain the role of BMAL1 in clock function. Circadian clocks in the brain thus possess a redundancy not found in peripheral clocks. We recently developed procedures for purifying native BMAL1 activator complexes from bovine cerebral cortex, making large-scale purification for biochemical and structural studies of brain circadian clock complexes practical. We find that BMAL1 activator complexes from brain are exclusively ~750 kDa in mass, much larger than a free heterodimer and similar to what we previously observed in the periphery. The complexes can be purified into two populations, one including BMAL1 and CLOCK (as in the periphery), the other BMAL1 and NPAS2 (not found in the periphery). Electron microscopy revealed the complexes to be discreet particles of ~20 nm in diameter, consistent with the estimated mass. The composition of these complexes is unknown; as expected of activator complexes, they do not appear to include circadian negative feedback proteins. Their mass is sufficient for roughly 10-12 proteins of average size. The full molecular identity and structure of the circadian clock transcriptional activators are thus not yet known, a fundamental gap in our understanding. The goal of this application is to analyze the composition of the two BMAL1 complexes purified from brain and develop procedures for determining their three-dimensional structures by cryo-electron microscopy. If successful, the project offers to deepen our knowledge of circadian clock transcription, provide potential new drug targets for circadian therapeutics, and reveal differences between brain and peripheral clocks. Advances in understanding the circadian clock are important for our knowledge of behavior and physiology, as well as for human health and disease. Clock defects produce disrupted sleep-wake cycles, metabolic syndrome, and increased cancer incidence and are linked to bipolar disorder. The proposed investigation aims to provide fundamental insights into the biological timing system essential for health.
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