Our long term goal is to describe in the language of genetics and biochemistry the feedback cycles and pathways that comprise intracellular circadian systems - how they work, how they are synchronized with the environment, and how time information generated by them is used to regulate the behavior of cells. This proposal focuses on the model system Neurospora, as well as on the mouse and mammalian cell lines, to understand the paradigms underlying circadian control of cell physiology and metabolism. We also continue a longstanding effort aimed at understanding circadian photoreception and photobiology in Neurospora and use this to break new ground on a salient fungal pathogen. Our work has three foci. One Focus anticipates a global analysis and description of the circadian output network in Neurospora, using RNA sequencing, chromatin immunoprecipitation, and bioinformatics to describe the regulatory hierarchy governing circadian regulation of transcription in a cell. Then, using the exceptional background of biochemical genetics available in Neurospora, we will track the metabolic activities carried out by the proteins encoded by clock-controlled genes, and as foci of clock-controlled activities emerge, we will begin to lay the spectrum of clock-controlled processes on the Neurospora metabolic map to see how the clock regulates metabolism and physiology. In the second Focus, we will apply our knowledge of circadian output pathways to mammalian cells, using RNA sequencing to determine the circadian profile of clock-controlled genes in adipocytes and in macrophages from wt and RIP140 knockout mice. We will use RNA-seq to characterize the transcriptomes of WT and mutant cells and use chromatin immunoprecipitation to begin to dissect the role of this co-activator/co-repressor in the circadian biology of these important cell types. These experiments will probe the significance of circadian regulation to fat metabolism and to immune function in a mammal, with the hope of gaining insights into the incidence in humans of diabetes, metabolic syndrome, time-of-day differences in immune function. A third Focus is in photobiology. We will explore the mechanism whereby light activates the principal fungal photoreceptor, and extend analysis of photobiology in the important fungal pathogen Aspergillus fumigatus. We envision and will test a way to exploit our understanding of fungal photobiology to enable a new treatment for hundreds of thousands of patients with aspergillosis. These projects are complementary and mutually enriching in that they each rely on genetic and molecular techniques to dissect, and ultimately to understand, the response of cells to their environment and the organization of eukaryotic cells as a function of time.
Biological clocks work in all cells of the human body to regulate metabolism. By studying cells of mammals, as well as cells of a fungus, we can understand how clock control works, how jet lag happens, and how clock malfunction leads to diseases like diabetes and mental illness.
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