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 Indiana Clinical and Translational Sciences Institute, the UND Eck Institute for Global Health, the UND Center for Rare and Neglected Diseases, and the University of Notre Dame, and previously by the Royal Society, the Wellcome Trust, the NIMH.
- Aaron Sheppard, Postdoctoral researcher
- Maricela Robles Murguia, Research associate
- Peng Zhou, Graduate student
- Sandy Duffield, Technician
- Alyssa Hummel, Undergraduate student
- John Werner, Undergraduate student
- David Lee, Undergraduate student
- Gary George, Undergraduate student
- Erin Clark, Undergraduate student
- Emma Cooper, Undergraduate student
- Dominic Acri, Undergraduate student
- Radoslaw Nabrzyski, Undergraduate student
- Anthony Nguyen, Undergraduate student
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.
- Suthat Liangpunsakul, Indiana University School of Medicine
- X. Charlie Dong, Indiana University School of Medicine
- 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, Faculty of Science & Technology, University of Westminster
- Frank Collins, University of Notre Dame
- W. Matthew Leevy, University of Notre Dame
- Robert Striker, Department of Medicine, University of Wisconsin
- Matthew Champion, University of Notre Dame
- Zain Syed, University of Notre Dame
Sheppard A.D., Rund S.S.C., George G.F., Clark E., Acri D., Duffield G.E. (2017) Light manipulation of mosquito behavior: acute and sustained photic suppression of biting in the Anopheles gambiae malaria mosquito. Parasites and Vectors 10:255.
Duffield G.E. (2016) ‘Understanding the genes that make our circadian clocks tick’. The Conversation (online media outlet), 4 November, 2016, https://theconversation.com/understanding-the-genes-that-make-our-circadian-clocks-tick-67356.
Rund, S.S.C., Yoo, B., Alam, C.D.C., Green, T., Stephens, M.T., Zeng, E., Sheppard, A.D., George, G.F., Duffield, G.E., Milenković, T., Pfrender, M.E. (2016) Genome-wide profiling of 24 hr diel rhythmicity in the water flea, Daphnia pulex: Network analysis reveals additional rhythmic gene expression and enhances functional gene annotation. BMC Genomics 17:653.
Zhou, P. Robles-Murguia M., Mathew D., and Duffield G.E. (2016) Impaired Thermogenesis and a Molecular Signature for Brown Adipose Tissue in Id2 Null Mice. Journal of Diabetes Research, vol. 2016, Article ID 6785948, 11 pages.
Champion M.M., Sheppard A.D., Rund S.S.C., Freed S.A., O’Tousa J.E. and Duffield G.E. (2015) Qualitative and quantitative proteomics methods for the analysis of the Anopheles gambiae mosquito proteome. Invited book chapter for ‘Short views on Insect Genomics and Proteomics: Insect Proteomics, Volume 2’ (Editors: T. Adeniran, R. Chandrasekar and M. Goldsmith), within the ‘Entomology in Focus’ book series (Editor: F.L. Cônsoli) (Springer publishers).
Zhou P., Werner J., Lee D., Sheppard A.D., Liangpunsakul S. and Duffield G.E. (2015) A dissociation between diurnal cycles in locomotor activity, feeding behavior and hepatic PERIOD2 expression in chronic alcohol-fed mice. Alcohol, 49:399-408.
Leming M.T., Rund S.S.C., Behura S.K., Duffield G.E., O’Tousa J.E. (2014) A database of circadian and diel rhythmic gene expression in the yellow fever mosquito Aedes aegypti. BMC Genomics 15:1128.
Metzinger M., Miramontes B., Zhou P., Liu Y., Peng J., Chapman S., Sun L., Sasser T.A., Duffield G.E., Stack M.S., Leevy W.M. (2014) Correlation of X-ray Computed Tomography with Quantitative Nuclear Magnetic Resonance (QMR) Methods for Pre-Clinical Measurement of Adipose and Lean Tissues in Living Mice. Sensors, 14: 18526-18542.
Zhou P., Hummel A.D., Pywell C.M., Dong X.C. and Duffield G.E. (2014) High fat diet rescues disturbances to metabolic homeostasis and survival in the Id2 null mouse in a sex-specific manner. Biochemical and Biophysical Research Communications 451: 374-381.
Balmert N.J., Rund S.S.C., Ghazi J.P., Zhou P., Duffield G.E. (2014) Time-of-day specific changes in metabolic detoxification and insecticide resistance in the malaria mosquito Anopheles gambiae. Journal of Insect Physiology 64:30-39 [published ahead of print 2014 March 11].
Zhou P., Ross R.A., Pywell C., Liangpunsakul S. and Duffield G.E. (2014) Disturbances to the murine hepatic circadian clock in alcohol-induced hepatic steatosis. Scientific Reports (Nature publishing) 4: 3725.
Mathew D., Zhou P., Pywell C.M., Van der Veen D.R., Shao J., Xi Y., Bonar N.A., Hummel A., Chapman S., Leevy W.M, Duffield G.E. (2013) Ablation of the Id2 gene results in altered circadian feeding behavior, and sex-specific enhancement of insulin sensitivity and elevated glucose uptake in skeletal muscle and brown adipose tissue. PLoS ONE 8: e73064.
Jagannath A., Butler R., Godinho S.I.H., Couch Y., Brown L.A., Vasudevan S.R., Flanagan K.C., Anthony D.C., Churchill G.C., Wood M.J., Steiner, G., Ebeling M., Hossbach M., Wettstein J.G., Duffield G.E., Gatti S., Hankins M.W., Foster R.G., Peirson S.N. (2013) The CRTC1-SIK1 pathway regulates entrainment of the circadian clock. Cell 154: 1100-1111.
Rund S.C., Bonar N.A., Champion M.M., Ghazi J.P., Houk C., Leming M.T., Syed Z., Duffield G.E. (2013) Daily rhythms in antennal protein and olfactory sensitivity in the malaria mosquito Anopheles gambiae. Scientific Reports (Nature publishing) 3: 2494.
Keating J.A., Bhattacharya D., Rund S.C., Hoover S., Dasgupta R., Lee S.J., Duffield, G.E., Striker R. (2013) Mosquito protein kinase G phosphorylates flavivirus NS5 and alters flight behavior in Aedes aegypti and Anopheles gambiae. Vector-Borne and Zoonotic Diseases 13: 590-600.
Rund, S.C., Gentile J.E. and Duffield, G.E. (2013) Extensive circadian and light regulation of the transcriptome in the malaria mosquito Anopheles gambiae. BMC Genomics 14: 218 [Epub ahead of print, April 3]
Rund S.S.C., Lee S.J., Bush B.R. and Duffield G.E. (2012) Strain- and sex-specific differences in daily flight activity and the circadian clock of Anopheles gambiae mosquitoes. Journal of Insect Physiology 58: 1609-19 [published ahead of print 2012 October 12].
Van der Veen D.R., Shao J., Xi Y., Li L. and Duffield G.E. (2012) Cardiac atrial circadian rhythms in PER2::LUCIFERASE and per1:luc mice: Amplitude and phase responses to glucocorticoid signaling and medium treatment. PLoS ONE 7: e47692.
Van der Veen D.R., Shao J., Chapman S., Leevy W.M. and Duffield G.E. (2012) A diurnal rhythm in glucose uptake in brown adipose tissue revealed by in vivo PET-FDG imaging. Obesity 20: 1527-9.
Duffield G.E., Mikkelsen, J.D. and Ebling F.J.P. (2012) Conserved Expression of Glutamate NMDA Receptor 1 Subunit Splice Variants during the Development of the Siberian hamster Suprachiasmatic nucleus. PLoS ONE 7: e37496.
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.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.
- Researchers use light to manipulate mosquitoes
- Duffield and Whaley among faculty honored for undergraduate teaching
- New paper describes genetic connections between biological rhythms, timing of feeding, and insulin sensitivity
- NDIIF announces awards for best imaging publications
- Aaron Sheppard wins first place in Postdoctoral Scholars Research Symposium
- New study reveals links between alcoholic liver disease and the circadian clock
- Lieberman and Duffield receive Indiana CTSI 2011 Fall Core Pilot Grants
- Researchers identify new drug target for treating jet lag and shift work disorders
- Mosquitoes smell you better at night
- New study explores how dengue virus changes mosquito behavior
- New paper examines shifting gears in the circadian clock of the heart
- Notre Dame researchers provide new genetic information about the circadian rhythms of the malaria mosquito
- New paper describes connections between Circadian and metabolic systems
- Paper sheds new “light” on fascinating rhythms of the circadian clock