Temperature entrainment of the molecular circadian clock circuits in Drosophila.

Many organisms rely on internal circadian clocks to organize their biological functions in an optimal daily schedule. Disruption of circadian clock function has been associated with negative outcomes for health and well-being, for example, in relation to sleep disorders, diabetes, high blood pressure, and cancer. Circadian clocks have a paradoxical relationship with temperature. On one hand, they employ so-called temperature compensation mechanisms to reliably keep daily time over a range of temperatures, but on the other hand they synchronize to daily temperature cycles with remarkable sensitivity. Daily rhythms in body temperature, which are themselves controlled by a clock in the brain, are thought to play a major role in maintaining synchrony between the clocks in different tissues. The fruit fly Drosophila melanogaster is an excellent model system for studying the internal circadian clocks that animals and humans use for daily time keeping. While little is known about the mechanisms that allow circadian clocks to maintain these sophisticated temperature responses, the Drosophila period (per) gene and its mammalian counterparts are thought to play a key role in daily time keeping as well as temperature entrainment and temperature compensation. The proposed project focusses on the impact of the recently discovered temperature-driven induction of the Drosophila per gene on temperature synchronization and compensation of daily time keeping. The sequence elements and transcription factors involved in this process will be identified by genetically testing different segments of the per gene as well as different potential regulators, respectively. Next, the functional importance of this process will be defined by describing changes in molecular or behavioural circadian rhythms resulting from genetic disruption of the required regulatory sequences and transcription complexes.
Given the high degree of structural and functional similarity between the per genes of flies and mammals, discoveries made for the role of temperature-driven per expression in the regulation of behavioural and molecular rhythms in flies are also relevant to understanding the impact of per expression in humans and other mammals on circadian physiology.

Circadian clocks generate and organize daily rhythms in animal physiology and behaviour. While their periodicity is temperature-compensated, circadian clocks in both poikolithermic and homeothermic organisms do synchronize to temperature cycles. The conserved core circadian regulator PERIOD (PER) is key to both temperature compensation and temperature entrainment. The PI recently uncovered strong temperature-driven transcriptional regulation of the per gene in the representative circadian model organism Drosophila melanogaster. Given the importance of the daily PER expression profile in the circadian oscillator as well as the proposed role of mammalian Per2 in temperature entrainment the newly uncovered temperature-driven per transcription is hypothesized to contribute to temperature-dependent regulation of daily time keeping. It is the aim of this project to provide insight in the mechanisms underlying circadian temperature entrainment and compensation by elucidating the role of temperature-driven per transcription. To this aim the responsible cis-regulatory elements will be genetically mapped in the per gene and their ability to confer temperature-driven regulation to the native per gene as well as reporter constructs will be determined. In addition, transcription factors involved in this process will be identified by candidate gene tests and systematic genetic screens in flies as well as a comprehensive set of Yeast 1-Hybrid assays. Molecular and behavioural time keeping, temperature entrainment, and temperature compensation phenotypes resulting from the disruption of the cis and trans-acting mediators of temperature-driven per transcription will be determined by gene expression and locomotor activity assays. Ultimately, this project is expected to provide new insights in the mechanisms underlying temperature-dependent regulation of molecular and behavioural circadian rhythms in animals and humans involving the conserved core clock component PER.

1)The Academic Community (see Academic Beneficiaries)
Will benefit from implications from our work for the many biological processes and systems that are subject temperature-entrained circadian regulation. Circadian rhythms are pervasive in organisms as diverse as cyanobacteria and humans and modulate a wide variety of biological functions including gene expression, metabolism, photosynthesis, cell division, growth, detoxification, digestive function, respiratory function, cardiovascular function, muscle strength, cognitive function, development, mating behaviour, and sleep. The impact of our research, therefore, extends well beyond our colleagues working on circadian rhythms or other aspects of Drosophila biology. The training of the postdoctoral researcher will also contribute to preparing the next generation of prinicipal investigators.
2)The Non-Scientific Community
Will benefit from educational outreach efforts pursued in connection with our work. The scientific relevance and potential applications from our work will be communicated to secondary school students, non-science university students, and the public at large. We will actively pursue mechanisms to reach these target audiences using a number of media and venues.
3)The Commercial Private Sector
Will benefit from the information from our work relevant to the diagnosis and treatment of human and animal disease as well as the development of new strategies for insect control.
a)The Biopharmaceutical Industry
Will benefit from new insights in the conserved role of PERIOD (PER) and its relevance for drug design. A number of PER-associated proteins are drug targets of commercial interest. Mammalian CRY1 and CRY2, which cooperate with PER1,2,3 in the inhibition of CLOCK/BMAL1 complexes are specifically affected by the component KL001, which has a potential application in the treatment of diabetes (Hirota, T. et al. Science 337:1094, 2012). Pharmacological inhibitors of Casein Kinase 1delta/epsilon (CK1d/e), which binds and regulates PER in both flies and mammals, are known to produce strong effects on circadian period length (Isojima, Y. et al. PNAS 106:15744, 2009; Hirota, T. et al. PNAS 105:20746, 2008; Hirota, T. et al. PLoS Biol. 2010, 8:e1000559, 2010; Meng, Q.J. et al. PNAS107:15240 2010). In fact, a number of kinase inhibitors in clinical trials for cancer treatment were found to affect both in vitro CK1d/e kinase activity and circadian period length in tissue culture (Isojima, Y. et al. PNAS 106:15744, 2009; Hirota, T. et al. PNAS 105:20746, 2008) suggesting that these drugs may have unintended side effects on circadian clocks. In addition, compounds affecting two other PER kinases with roles in the circadian time keeping of both flies and mammals (GSK3-beta and Casein Kinase 2) also affect molecular circadian rhythms in tissue culture. Our identification of the impact of temperature-driven regulation of PER on circadian temperature entrainment and compensation in Drosophila will yield testable hypotheses regarding analogous mechanisms in mammals and the involvement of known drug targets.
b)The Insect Control Industry
Will benefit from implications of our research relevant to strategies for control of harmful insects that target circadian control of insect development and behaviour. One of the conserved functions of the circadian clock is its control of the rhythmic expression of detoxifying enzymes, including those involved in regulating insecticide resistance. The clock-controlled transcription factor PDP1, which acts downstream of PER plays an important role in this process (Wijnen, H. & Young, M.W. Annu Rev Genet 40:409, 2006, Beaver, L. M. et al. Toxicol Sci 115:513, 2010. Hooven, L. A., et al., PLoS One 4:e6469, 2009.). Our experiments will clarify the role of temperature-driven transcription of per in the generation of temperature-modulated circadian expression throughout the adult fly head, which includes tissues relevant to detoxification.