Regulatory mechanisms of PDF neuropeptide production in the Drosophila clock neurons

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PROJECT SUMMARY Construction of functional nervous system in the central nervous system (CNS) requires elaborate connectivity of immensely heterogeneous neuronal cell types. One such neural circuit is dedicated to control various biological rhythms with approximately 24-hour periodicity, termed circadian rhythms. Disruption of the rhythms is closely associated with various health-related problems such as sleep disorders, jet-lag, job-performance associated with shifted works, autoimmune disease, cancer, and Alzheimer’s disease. The cellular and molecular bases of the endogenous clock have been well studied particularly in Drosophila. Approximately 150 neurons in the adult Drosophila CNS form a neural circuit regulating the circadian rhythmic behavior and physiology. These clock neurons intimately communicate with each other via secretory factors from these neurons. A major clock messenger molecule is a neuropeptide, pigment dispersing factor (PDF), which is produced by a group of major clock neurons, s- LNvs. Despite these studies, how Pdf gene expression is regulated and how the PDF precursor molecules are processed to secrete functionally mature PDF are not well understood. These regulatory mechanisms are certainly important for timely production of PDF to regulate circadian rhythms. From our studies on the transcriptional mechanism of Pdf, we have recently reported a 35-bp Pdf regulatory element (PRE) that is essential for the Pdf expression. To gain further insight into the mechanisms underlying PDF production in the clock neurons, we propose the following aims: (1) Functional dissection of PRE in vivo. Using CRISPR/Cas9 and CRISPR interference (CRISPRi) tools, we will demonstrate the endogenous role of PRE in vivo and determine s-LNv-specific element within PRE. (2) Screening Pdf- regulating transcription factors. We will employ both gain-of-function and loss-of-function strategies to identify potential candidates that bind to PRE and thus regulate Pdf expression. (3) Clock-controlled processing of PDF precursor. Functionally mature PDF is part of a large precursor, which is predicted to be processed via proteolytic cleavage and modified by C-terminal amidation. Our previous data suggest that such post-translational events might be clock-controlled to release PDF. We will develop a novel immunological as well as various genetic and transgenic tools to study the mechanisms of PDF maturational process. (4) Characterization of a new Clock (Clk) mutant allele. Clk gene is a central clock factor that also regulates Pdf expression indirectly. Recently we have found a new arrhythmic Clk mutant allele, which we call tentatively no-time (Clknot). Molecular characterization of this new mutant allele will shed more light on the functional aspects of CLK protein.