We have been studying the molecular control of circadian behavioral rhythms using Drosophila as a model system. Homologues of genes initially characterized in the fly, have now been linked to the control of hythmic behavior and physiology in vertebrates, including fish, frogs, mice and humans. A central component of the fly clock is a feedback circuit in which two clock proteins, PERIOD (PER) and TIMELESS (TIM), repress their own transcription. Temporal delays in this feedback promote oscillatory gene expression. We have recently discovered novel cellular features controlling one such delay. Additional studies have identifed new genes and proteins affecting periodicity of the circadian clock. In this proposal we will examine the following: (1) PER and TIM appear to be physically modified in response to their interaction in the cytoplasm, allowing their subsequent, independent nuclear accumulation. We will identify and characterized modifications of these proteins that are associated with this regulation. (2) We will determine whether a newly discovered, PER/TIM cytoplasmic interval timer contributes to temperature compensation of the circadian clock. (3) We will conduct a high-throughput screen for new genes and proteins regulating the timed nuclear accumulation of PER and TIM in cultured cells. (4) A locomotor activity screen involving several hundred transgenic RNAi stocks has shown that reduction of a specific karyopherin substantially lengthens the period of the fly clock. The molecular pathway underlying this protein's contribution to rhythmicity will be explored in flies and S2 cells. (5) Cryptochrome (CRY) has a key role in the light-dependent degradation of TIM. We have produced new mutations that affect physical interactions of the CRY C-terminal tail with the CRY photolyase homology domain (PHD). Preliminary studies indicate that some of these mutations alter CRY stability only on exposure to light. We will determine whether light induces dissociation of the CRY C-terminal tail and the PHD.
Candidate gene approaches, originating in the forward genetic screens of Drosophila, allowed mutant orthologs of human PERIOD protein and casein kinase 1 to be connected to inborn errors of sleep. The early functional studies of these genes and proteins in Drosophila have also been used as the basis for exploring specific mechanisms underlying aberrant patterns of human sleep. We believe our proposed genetic, biophysical, and biochemical studies of Drosophila's circadian clock will continue to reveal new principles of organization and function that promote an understanding of human circadian rhythms.
|Top, Deniz; Young, Michael W (2017) Coordination between Differentially Regulated Circadian Clocks Generates Rhythmic Behavior. Cold Spring Harb Perspect Biol :|
|Garaulet, Daniel L; Sun, Kailiang; Li, Wanhe et al. (2016) miR-124 Regulates Diverse Aspects of Rhythmic Behavior in Drosophila. J Neurosci 36:3414-21|
|Ganguly, Abir; Manahan, Craig C; Top, Deniz et al. (2016) Changes in active site histidine hydrogen bonding trigger cryptochrome activation. Proc Natl Acad Sci U S A 113:10073-8|
|Top, Deniz; Harms, Emily; Syed, Sheyum et al. (2016) GSK-3 and CK2 Kinases Converge on Timeless to Regulate the Master Clock. Cell Rep 16:357-367|
|Axelrod, Sofia; Saez, Lino; Young, Michael W (2015) Studying circadian rhythm and sleep using genetic screens in Drosophila. Methods Enzymol 551:3-27|
|Jang, A Reum; Moravcevic, Katarina; Saez, Lino et al. (2015) Drosophila TIM binds importin ?1, and acts as an adapter to transport PER to the nucleus. PLoS Genet 11:e1004974|
|Kidd, Philip B; Young, Michael W; Siggia, Eric D (2015) Temperature compensation and temperature sensation in the circadian clock. Proc Natl Acad Sci U S A 112:E6284-92|
|Crane, Brian R; Young, Michael W (2014) Interactive features of proteins composing eukaryotic circadian clocks. Annu Rev Biochem 83:191-219|
|Vaidya, Anand T; Top, Deniz; Manahan, Craig C et al. (2013) Flavin reduction activates Drosophila cryptochrome. Proc Natl Acad Sci U S A 110:20455-60|
|Levy, Colin; Zoltowski, Brian D; Jones, Alex R et al. (2013) Updated structure of Drosophila cryptochrome. Nature 495:E3-4|
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