Molecular mechanisms of mammalian circadian clock function

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Abstract Circadian clocks throughout the body drive rhythmic expression of thousands of genes, resulting in rhythms in biochemistry, physiology and behavior. Disruption of circadian clocks through genetics or environmental perturbations such as jet lag or shift-work, can have profound negative consequences and has been linked to obesity, diabetes, cancer, cardiovascular disease and mental illness. Our work is focused generally on understanding the detailed molecular mechanisms of the mammalian circadian clock machinery and the mechanisms by which these clocks control rhythmic gene expression. According to the current model, the core part of this clock mechanism is a negative feedback loop whereby the transcription factor heterodimer CLOCK/BMAL1 drives transcription of the “clock” proteins PERIOD (PER) 1, PER 2, CRYPTOCHROME (CRY) 1 and CRY 2 which interact with each other to repress the activity of CLOCK/BMAL1, and thus their own synthesis. We have solved crystal structures for the CLOCK/BMAL1 and CRY2/PER2 complexes and these data have allowed the identification of evolutionarily conserved functional domains throughout the proteins and revealed additional insights into the mechanisms by which these proteins operate and set the circadian period. Over the next five years, we will expand on this information to determine the atomic details of how this clock keeps time. The roles of these core circadian clock transcription factors in driving rhythmic transcription is well-documented, but recent data have demonstrated that post-transcriptional control, although much less well understood, is also critical for normal rhythmic protein expression profiles. One type of post-transcriptional control is regulation of mRNA poly(A) tail length, which impacts the stability and translational regulation of mRNA. We have identified hundreds of mouse liver mRNAs that exhibit robust circadian rhythms in the length of their poly(A) tails. In many of these cases, the rhythmic tail lengths are the result of rhythmic cytoplasmic polyadenylation and deadenylation rhythms and many components of the cytoplasmic polyadenylation and deadenylation machinery are themselves under circadian control. Furthermore, the rhythmic poly(A) tails are closely correlated with the rhythmic protein expression. Therefore, the circadian clock regulates dynamic polyadenylation status of many mRNAs that can drive rhythmic protein expression independent of the steady-state levels of the message. Nocturnin is a robustly rhythmic protein that removes poly(A) tails from mRNAs. We have shown that loss of this gene in mice causes resistance to diet-induced obesity and altered rhythms in cholesterol and triglyceride metabolism, implicating it as an important circadian post-transcriptional mediator. Over the next five years, we will focus on identifying the mRNA substrates of Nocturnin both in the cytosol and in the mitochondria.