Essentially all organisms use circadian clocks to control daily rhythms in physiology, metabolism and behavior. These clocks are of great clinical importance since their dysfunction can lead to sleep disorders and certain forms of depression. The identification and analysis of clock genes in Drosophila revealed that the circadian timekeeping mechanism is comprised of two interlocked transcriptional feedback loops. CLOCK (CLK)-CYCLE (CYC) heterodimers bind E-box elements to activate feedback regulators period (per), timeless (tim), vrille (vri) and Par domain protein 1e (Pdple), which control circadian transcription. VRI and PDP1e bind V/P-boxes to control rhythms in Clk transcription that peak near dawn, whereas PER and TIM inhibit CLK-CYC activity to control rhythms in E-box dependent clock gene transcription that peak near dusk. Even though CLK-CYC dependent activation and PER-TIM dependent inhibition directly or indirectly control all circadian transcription, how PER-TIM feeds back to inhibit CLK-CYC transcriptional activity is not well understood. We find that CLK-CYC binds E-boxes in vivo only at times when their target genes are transcribed, which differs from the analogous E-box activators in mammals that bind constitutively or bind primarily when target gene transcription is inhibited. This result suggests that PER-TIM functions to inhibit E- box binding by CLK-CYC, thus enabling us to determine which E-boxes are important for CLK-CYC dependent transcription and which genes are directly controlled by CLK-CYC. We also find that CLK phosphorylation is high when CLK-CYC forms a transcriptionally inactive nuclear complex with PER-TIM and DOUBLE-TIME (DBT) kinase, and is low when CLK-CYC binds E-boxes and activates transcription. This, together with results showing CLK phosphorylation is PER dependent and DBT destabilizes CLK, suggests that interactions between CLK and PER-TIM-DBT produce a rhythm in CLK phosphorylation that regulates CLK stability and CLK-CYC transcriptional activity. In this proposal, we will define the molecular mechanisms that control rhythmic transcription by determining how (1) rhythms in CLK-CYC binding activity are regulated, (2) E-boxes contribute to rhythmic transcription within and downstream of the feedback loops, and (3) CLK phosphorylation affects CLK stability and CLK-CYC activity. Since circadian timekeeping mechanismsfrom flies and mammals are well conserved, what we learn here will be relevant to the situation in mammals.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS052854-04
Application #
7576901
Study Section
Special Emphasis Panel (ZRG1-NCF (09))
Program Officer
Mitler, Merrill
Project Start
2006-04-01
Project End
2010-08-15
Budget Start
2009-04-01
Budget End
2010-08-15
Support Year
4
Fiscal Year
2009
Total Cost
$286,094
Indirect Cost
Name
Texas A&M University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
078592789
City
College Station
State
TX
Country
United States
Zip Code
77845
Agrawal, Parul; Hardin, Paul E (2016) An RNAi Screen To Identify Protein Phosphatases That Function Within the Drosophila Circadian Clock. G3 (Bethesda) 6:4227-4238
Agrawal, Parul; Hardin, Paul E (2016) The Drosophila Receptor Protein Tyrosine Phosphatase LAR Is Required for Development of Circadian Pacemaker Neuron Processes That Support Rhythmic Activity in Constant Darkness But Not during Light/Dark Cycles. J Neurosci 36:3860-70
Lee, Euna; Jeong, Eun Hee; Jeong, Hyun-Jeong et al. (2014) Phosphorylation of a central clock transcription factor is required for thermal but not photic entrainment. PLoS Genet 10:e1004545
Menet, Jerome S; Hardin, Paul E (2014) Circadian clocks: the tissue is the issue. Curr Biol 24:R25-R27
Mahesh, Guruswamy; Jeong, EunHee; Ng, Fanny S et al. (2014) Phosphorylation of the transcription activator CLOCK regulates progression through a ? 24-h feedback loop to influence the circadian period in Drosophila. J Biol Chem 289:19681-93
Hardin, Paul E; Panda, Satchidananda (2013) Circadian timekeeping and output mechanisms in animals. Curr Opin Neurobiol 23:724-31
Kaneko, Haruna; Head, Lauren M; Ling, Jinli et al. (2012) Circadian rhythm of temperature preference and its neural control in Drosophila. Curr Biol 22:1851-7
Hardin, Paul E (2011) Molecular genetic analysis of circadian timekeeping in Drosophila. Adv Genet 74:141-73
Yu, Wangjie; Houl, Jerry H; Hardin, Paul E (2011) NEMO kinase contributes to core period determination by slowing the pace of the Drosophila circadian oscillator. Curr Biol 21:756-61
Yu, Wangjie; Zheng, Hao; Price, Jeffrey L et al. (2009) DOUBLETIME plays a noncatalytic role to mediate CLOCK phosphorylation and repress CLOCK-dependent transcription within the Drosophila circadian clock. Mol Cell Biol 29:1452-8

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