Identification and Analysis of Circadian Clock-Controlled Genes

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Project Summary This is a proposal for a one year terminal Supplement to my existing MIRA grant for the purposes of finishing off, where possible, remaining activities and transitioning personnel in anticipation of my exiting full time research. Despite my plans, this transitioning has been made very difficult due to the pandemic. For context, my long term goal has been 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 for a one year Supplement 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. The goal is to transition ongoing work to a state where it can be published or handed off in an orderly manner for completion. One Focus anticipates completion of a global analysis and description of the light-response network in Neurospora, using RNA sequencing, chromatin immunoprecipitation, and bioinformatics to describe the regulatory hierarchy governing the transcriptional response of the cell to light. This response is initiated by the same transcription factor that initiates control circadian response, and description of the light pathway. I believe this is achievable in the final year, is an important first step to describing the circadian transcriptional control network. This work is a restricted subset of the research approach originally anticipated. 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 from wt and RIP140 knockout mice. We will use RNA-seq to characterize the transcriptomes of WT and mutant cells and use chromatin immunoprecipitationto 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 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. This will complete an important step in this analysis and is achievable in a final year. 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.