Molecular biology of circadian rhythms in mammals and mosquito vectors. Clock control of metabolism.
Giles Duffield
e-mail Eck Institute for Global Health website
Assistant Professor
Ph.D., University of Cambridge, 1998
Postdoctoral and Faculty Research (Wellcome Trust Research Fellow, Royal Society University Research Fellow), Dartmouth Medical School and Imperial College London
Telephone: 574-631-1834
Fax: 574-631-7413
The circadian clock regulates 24-hour endogenous rhythms in gene expression, biochemistry, physiology and behavior of all eukaryotic organisms. This clock is based on a cell autonomous system comprised of transcriptional-translational feedback loops. My laboratory is focused on understanding the molecular basis for the circadian clock in both vertebrate (mouse and mammalian tissue culture) and invertebrate (mosquito) animals. Circadian clock biology is relevant to human health. Dysfunction of the circadian clock underlies several disease states, including Seasonal Affective Disorder, and sleep and metabolic disorders associated with shift-work, including obesity and diabetes. Understanding of the circadian system within the African malaria vector, such as Anopheles gambiae, may allow generation of improved insect control and malaria transmission controls strategies. The lab is interested in elucidating the molecular basis of the circadian clock in the mouse and insects using a range of traditional and state of the art molecular, cellular and behavioral approaches. Our studies are supported by the National Institute of General Medical Sciences (NIGMS), the American Heart Association, the Eck Institute for Global Health and the University of Notre Dame, and previously by the Royal Society, the Wellcome Trust, the NIMH.
Role of Inhibitor of DNA binding genes in the Regulation of the Mammalian Circadian Clock. 
The molecular circadian clock consists of an autoregulatory transcriptional-translational feedback loop composed of positive and negative regulators. Work over the last 14 years has identified at least nine such components, but additional genes and modifiers are being identified. In addition, most tissues of the body harbor cell-autonomous circadian clocks. One such additional gene, identified in my laboratory using a DNA microarray screen, is the transcriptional inhibitor Inhibitor of DNA-binding 2 (ID2). It is rhythmically expressed in the master clock structure in the hypothalamic brain known as the suprachiasmatic nucleus (SCN), and throughout the body in various peripheral tissues (e.g. heart and liver). Current studies are to evaluate the role of genes such as Id2 in the organization of the central oscillator, and to identify novel molecules relevant to cellular clock function. We are also interested in understanding how light resets the molecular clock (input), and how the clock regulates down-stream clock-controlled genes (i.e. output, hands of the clock). The lab is using continuous activity monitoring to identify behavioral phenotypes in transgenic mouse models (e.g. Id2 knockout mice) that are maintained under a variety of photocycle conditions, and exposed to artificial time-zone changes and acute light/pharmacologic/behavioral treatments. Results thus far have revealed that in the absence of the Id2 gene, mice adapt to large time-zone changes (e.g. mimicking a flight from Berlin to Los Angeles) more rapidly than wildtype individuals. We are also using real-time monitoring of clock gene expression in tissues and cells derived from transgenic mice that express Firefly luciferase in a rhythmic manner. We are using DNA microarray and real-time quantitative RT-PCR analyses to identify and characterize clock regulated genes in brain and peripheral tissues (e.g. liver and heart); tissue culture of immortalized fibroblasts that exhibit circadian oscillations in gene expression as a model of the in vivo rodent circadian clock; protein interaction analyses (e.g. western blot, co-immunoprecipitation, 2-hybrid, immunofluorescence) to understand interaction dynamics between clock related proteins; and traditional neuroanatomical techniques (e.g. in situ hybridization, immunohistochemistry, neuronal track tracing) to characterize clock gene function in the brain. Furthermore, we have begun to utilize Drosophila fruit fly as a tractable system in which to examine the role of Id genes further within the circadian system.
Role of Id2 in metabolic regulation.
We are interested in the relationship between the circadian clock and lipid and glucose metabolism. We have demonstrated that Id2 null mice exhibit a metabolic phenotype characterized by low lipid storage (lean body form, low quantity of white adipose tissue, and reduced lipid levels in liver) and disturbances in rhythms in genes expressed in the liver that are associated with daily changes in lipid and glucose availability and utilization. Current studies are to evaluate the role of genes such as Id2 in the pathways through which the circadian clock regulates 24 hr rhythms in metabolic function.
Circadian Genomics of Anopheles gambiae.
The molecular basis for circadian rhythms has been investigated in model organisms such as the mouse and Drosophila, but little is known of circadian regulation in disease vector species, such as the Anopheles mosquito (malaria vector). The mosquito exhibits a wealth of circadian behavior, such as feeding and breeding activity, but almost nothing is know of how the circadian system in this insect family are comprised and how the core molecular clock components regulate physiology and behavior. This research examines the circadian clock of Anopheles gambiae, a vector of the malaria parasite in Africa. We are interested in 1) understanding how the circadian clock is comprised at a molecular and cellular level; and 2) defining the circadian control of the transcriptome and to elucidate pathways that interconnect with overt rhythmic aspects of the species behavior and physiology. Research in the laboratory emphasizes techniques of molecular biology, genetics, and genomics, including high density DNA microarray analysis. We have discovered that genes associated with processes of metabolism, detoxification and sensory modalitities of vision and olfaction are under rhythmic control. Characterization of the circadian regulation of the mosquito transcriptome will allow us to better understand both insecticide resistance in this species, as well as generate improved understanding of temporal gated behavior such as time of blood feeding. Furthermore, examination of A. gambiae sub-species that exhibit differences in their circadian behavior, physiological responses and resistance to insecticides, will allow us to identify specific underlying molecular pathways that correlate with the variants observed in the overt rhythms.
Anopheles gambiae mosquito gene expression data (www.nd.edu/~bioclock)
"Cartoons for Conservation" web link is http://www.alanhesse.co.uk/
Research Personnel
Deepa Mathew, Postdoctoral researcher
Sam Rund, Graduate Student
Jinping Shao, Graduate Student
Peng Zhou, Graduate Student
Yang Xi, Graduate Student
Nicolle Bonar, Research Visitor
John Ghazi, undergraduate student
Brian Bush, undergraduate student
Cameron Pywell, undergraduate student
Nate Balmert, undergraduate student
Sam Lee, undergraduate student
Duffield lab, December 2011
Former Personnel
Daan van der Veen, Postdoctoral researcher (now a Lecturer at University of Surrey, UK)
Sarah Ward, Graduate Student (graduated with Ph.D.; now a Postdoctoral researcher at Northwestern University)
Maricela Robles Murguia, Research Technician (now a Research Assistant at Kansas State University)
Tim Hou, Research Associate (now a graduate student at Texas A&M University)
Kevin Flanagan, undergraduate student (now a graduate student at Washington University in St. Louis)
Shanik Fernando, undergraduate student (now a medical student at Vanderbilt University)
Kathleen McDonald, undergraduate student
Collaborators
- Mark Israel, Director, Norris Cotton Cancer Center, Dartmouth Hitchcock Medical Center
- Jay Dunlap and Jennifer Loros, Department of Genetics, Dartmouth Medical School
- Horacio de la Iglesia, Department of Biology, University of Washington
- Stuart Peirson, Department of Ophthalmology, University of Oxford
- Nelson Chong, Lecturer in Molecular Cardiology, Department of Cardiovascular Sciences, Leicester Medical School, University of Leicester
- Frank Collins, University of Notre Dame
- W. Matthew Leevy, University of Notre Dame
Selected Bibliography
Van der Veen D.R., Shao J., Chapman S., Leevy W.M. and Duffield G.E. (2012) A 24-hour temporal profile of in vivo brain and heart PET imaging reveals a nocturnal peak in brain 18F-fluorodeoxyglucose uptake. PLoS ONE 7: e31792.
Rund, S.C., Hou, T.Y., Ward, S.M., Frank H. Collins, F.H. and Duffield, G.E. (2011) Genome-wide profiling of diel and circadian gene expression of the malaria vector Anopheles gambiae. Proceedings of the National Academy of Sciences USA 108 (32): E421-E430 [published ahead of print June 29].
Ward, S.M., Fernando, S., Hou, T.Y. and Duffield, G.E. (2010) The transcriptional repressor ID2 can interact with the canonical clock components CLOCK and BMAL1 and mediate inhibitory effects on mPer1 expression. Journal of Biological Chemistry Dec 10; 285:38987-39000 [Epub 2010 Sep 22].
Hou, T., Ward, S.M., Murad J.M., Watson, N.P., Israel, M.A. and Duffield, G.E. (2009) Inhibitor of DNA binding 2 (ID2) is a rhythmically expressed transcriptional repressor required for circadian clock output in the mouse liver. Journal of Biological Chemistry Nov 13; 284:31735-45 [Epub 2009 Sep 9].
Duffield G.E., Robles-Murguia M., Watson N.P., Mantani A., Peirson S.N., Loros J.J., Israel M.A., Dunlap J.C. (2009) A role for the Id2 gene in regulating photic entrainment of the mammalian circadian system. Current Biology; 19:297-304.
Peirson, S.N., Butler, J.N., Duffield, G.E., Takher, S., Sharma, P. and Foster, R.G. (2006). Comparison of clock gene expression in SCN, retina, heart and liver of mice. Biochemical and Biophysical Research Communications; 351:800-807.
Duffield, G., Loros, J.J., and Dunlap, J.D. (2005) Analysis of circadian output rhythms of gene expression in neurospora and mammalian cells in culture. Methods Enzymology 393:315-341. Invited paper for edition on Circadian Rhythms.
Duffield, G.E. (2003) DNA microarray analyses of circadian timing (Invited Review, ‘Young Investigator’s Perspectives’). Journal of Neuroendocrinology 15:991-1002.
Nowrousian, M., Duffield, G.E., Loros, J.J. and Dunlap, J.C. (2003) The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa. Genetics 164:923-933.
Duffield, G.E., Best, J.D., Meurers, B.H., Bittner, A., Loros, J.J. and Dunlap, J.C. (2002) Circadian programs of transcriptional activation, signaling, and protein turnover revealed by microarray analysis of mammalian cells. Current Biology 12:551-557, April 3 (cover article).
Duffield, G.E., McNulty, S. and Ebling, F.J.P. (1999) Anatomical and funcational characterization of a dopaminergic system in the suprachiasmatic nucleus of the neonatal Siberian hamster. Journal of Comparative Neurology 408:73-96.
von Gall, C., Duffield, G.E., Hastings, M.H., Kopp, M.D.A., Dehghani, F., Korf, H.-W., Stehle, J.H. (1998). CREB in the Mouse SCN: A Molecular Interface Coding the Phase-Adjusting Stimuli Light, Glutamate, PACAP, and Melatonin for Clockwork Access. Journal of Neuroscience 18: 10389-10397.
Duffield G.E., Hastings M.H. and Ebling F.J.P. (1998). Investigation into the regulation of the circadian system by dopamine and melatonin in the Siberian hamster (Phodopus sungorus). Journal of Neuroendocrinology 10: 871-884.
