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We are focusing our efforts on understanding 3 fundamental features of the circadian clock, using Neurospora crassa as a model organism:
1. How do circadian oscillators (the time keeping mechanism analogous to the gears and cogs of a watch) signal to regulate daily rhythms in gene expression and behavior (the hands of the clock)?
Using genetic approaches in Neurospora, we discovered that the clock regulates major signaling pathways, called MAPK pathways. These same signaling pathways are present in humans and are involved in stress responses, immune activity, and cell division. The circadian regulation of MAPK pathway activation provides a rationale for observations that deregulation of the clock contributes to cancer, and suggests new opportunities for treatment. Current projects involve determining the precise mechanism of this regulation, and the consequences of disrupting circadian control of the MAPK pathway. In addition, we are investigating clock regulation of the p38 MAPK pathway in mammalian cell lines.
In addition, in a large collaborative project, we used ChIP-seq to identify direct targets of oscillator components (the WCC) in order to understand the mechanisms by which clock components signal through circadian output pathways. We identified several transcription factors as targets of the WCC, and are currently using ChIP-seq, combined with RNA-seq, to identify the second tier targets. These high throughput genomics approaches will eventually lead to a network map of the circadian output pathways.
2. How complex is the circadian system and how do individual oscillators communicate with each other to coordinately regulate rhythmicity?
Using genetic approaches, we have discovered that similar to the mammalian clock, the clock mechanism in fungi is complex and is constructed using multiple circadian oscillators. In Neurospora, the oscillator that has been well described is called the FRQ/WCC oscillator. The FRQ/WCC oscillator regulates daily rhythms in development and gene expression. The FRQ/WCC oscillator comprises a negative feedback loop that takes a day to be completed. The WCC activates frq gene expression and FRQ protein negatively regulates the activity of the WCC, thereby shutting down its own expression. However, we found evidence for the existence of circadian oscillations in gene expression and development that don’t require a functional FRQ/WCC oscillator. We are currently determining the components of the oscillators and working to establish the rules that govern communication between them. This discovery provides a unique opportunity to learn how circadian oscillators in multicellular tissues talk to each other to coordinately regulate robust rhythms in the organism.
3. How is temperature compensation of the clock achieved?
A unique feature of the circadian clock is that it is relatively insensitive to different temperatures. This is unlike most biochemical reactions in which increased temperature increases the rate of the reaction. We, and others, have found that temperature compensation arises from the activity of conserved protein modifying enzymes (kinases) that act on the components of the oscillator. The kinases are themselves temperature-dependent. We are focusing on the PAS-domain kinase, PSK, which phosphorylates the WCC at high temperatures and regulates its activity in the circadian oscillator. Current projects involve identifying the sites of action for these modifying enzymes using biochemical approaches, and involve discovering new modifiers.