Circadian oscillators control daily rhythms in diverse organisms. In humans, abnormalities in the oscillator are associated with sleep disorders, cardiovascular disease, metabolic syndrome, and cancer. Unlike most biochemical processes that speed up as temperature rises, a universal property of the circadian clock is that it maintains nearly constant rate (period) at all physiological temperatures. This process, called temperature compensation (TC), is conserved in organisms that maintain their own body temperature. However, the mechanism of TC, and the impact of TC in circadian health is not known. In Neurospora, the organism in which temperature responses of the clock are best known, TC involves the activity of a conserved PAS kinase (PSK). PSK phosphorylates the positive-acting clock component WCC at high temperature. Cells that lack PSK have a clock that runs faster at high temperatures. Casein kinase 2 (CK2) phosphorylates and reduces the activity of the negative-acting clock component FRQ at high temperature, and cells lacking CK2 run slower at high temperatures. However, strains with defects in PSK and CK2 are fully compensated, consistent with observations that other factors modify the clock components. We propose to test, and refine, two competing hypotheses for TC. 1) An intrinsic temperature-dependent increase in the levels of the positive and negative clock components counter-balance each other to maintain consistent period. 2) TC is an emergent network in which period modifying enzymes alter the activities of the clock components to compensate for their observed increased levels at high temperature.
In Aim 1, we will test the model that PSK phosphorylation of the WCC, as opposed to some other target, is necessary for TC. PSK target sequences on WCC proteins will be determined using mass spectroscopy in wild type (WT) and PSK-deletion strains at low and high temperature at different times of the day. PSK phosphorylation sites will be mutated, and the strains examined for loss of TC. In the same experiment, other potential temperature-dependent modifications of the clock components in WT cells will be identified, and strains with mutations of the modifications sites examined for loss of TC.
In Aim 2, we will test the models for TC by identifying temperature-dependent period modifiers. Combinations of deletion mutations of the TC factors will be used to test the emerging network hypothesis. Selectively manipulating the levels of the WCC within strains (while maintaining rhythmicity) to change the relative balance of the clock components will test the intrinsic TC hypothesis.
In Aim 3, we will determine the mechanism regulating a temperature-dependent increase in clock proteins and PSK. Using whole genome ribosome profiling coupled with transcriptome analyses;we will test the hypothesis that a physiological temperature increase specifically affects translation efficiency of the clock components. Together these studies will have a major impact on our understanding of TC, a key conserved aspect of circadian clocks.

Public Health Relevance

Temperature compensation (TC) of period is unique and fundamental feature of all circadian clocks, but is not at all understood. Our investigation of TC, fueled by the discovery of a key molecule required for TC, has broad implications for understanding and treating human diseases associated with a defective clock.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Special Emphasis Panel (ZRG1-CB-R (02))
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Sesma, Michael A
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Texas A&M University
Schools of Arts and Sciences
College Station
United States
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Wu, Cheng; Dasgupta, Ananya; Shen, Lunda et al. (2018) The cell free protein synthesis system from the model filamentous fungus Neurospora crassa. Methods 137:11-19
Hughes, Michael E; Abruzzi, Katherine C; Allada, Ravi et al. (2017) Guidelines for Genome-Scale Analysis of Biological Rhythms. J Biol Rhythms 32:380-393
Lamb, Teresa M; Vickery, Justin; Bell-Pedersen, Deborah (2013) Regulation of gene expression in Neurospora crassa with a copper responsive promoter. G3 (Bethesda) 3:2273-80