Lee Laboratory Choogon Lee, Ph.D. Florida State University College of Medicine Dept. of Biomedical Sciences 1115 West Call Street Tallahassee, FL 32306-4300 Office: (850) 645-1478, MSR 2300-L Lab: (850) 645-1508, MSR 2310-N Dr. Lee's Faculty Profile
Research Interests
Research in my lab has focused on the molecular mechanism of mammalian circadian (≈ 24 hr or daily) rhythms. Circadian rhythms have been observed in nearly all organisms from cyanobacteria to humans. These rhythms are under the direct influence of environmental cues, most notably the day/night cycle, and by a genetically determined, endogenous clock called the “circadian clock”. Our own sleep/wake rhythm is the most familiar circadian rhythm, but there is circadian rhythmicity in many aspects of physiology including alertness, activity, hormone production and drug efficacy. These and other daily activities and physiological processes are under the control of the circadian clock. The circadian clock is cell-autonomous and ubiquitously present in most tissues. The mammalian circadian clock operates through a self-sustaining, transcriptional negative feedback loop, as found in most, if not all, organisms. In mammals, two basic-helix-loop-helix (bHLH)/PAS-containing transcription factors, CLOCK and BMAL1, constitute the positive elements. The CLOCK:BMAL1 heterodimer activates the transcription of the negative elements, Period (Per) and Cryptochrome (Cry) genes. There are three Per (Per1-3) genes and two Cry genes (Cry1 and Cry2). Based on biochemical and genetic studies, CRY protein plays a major role in this inhibition, while PER protein regulates the timing of inhibition by controlling accessibility (nuclear entry) of CRY protein to the CLOCK:BMAL1 protein complex. Casein kinase Ιє⁄δ (CKIє/δ) are kinases for PER protein (Figure 1). The regulation of PER protein by phosphorylation appears to be a key step for producing a normal 24-hour molecular clock. Indeed, recent evidence indicates that a mutation in a putative phosphorylation site of human PER2 causes a severe sleeping disorder known as familial advanced sleep phase syndrome. This was the first demonstration that a mutation in a clock gene underlies a human disorder.
Current Projects
Roles of CKIє/δ in the mammalian clock mechanism Two kinases believed to phosphorylate mPER are casein kinase Iє and Iδ (CKIє/δ). A number of studies suggest that CKIє/δ are likely to be important for circadian clock function. To study the circadian roles of these kinases more decisively, we generated novel transgenic mice in which CKIє/δ activities are disrupted by a dominant negative (DN) CKIє/δ. The DN-CKIє is expressed under the post-embryonically active promoter, the Albumin promoters. We used this approach because a complete knockout of CKIє/δ would likely be lethal, as the kinases play a role in Wnt signaling during development. By assessing molecular and behavioral perturbation caused by disruption of CKIє/δ, we should be able to deduce the roles of CKIє/δ in the mammalian clock. Roles of β-Trcp, a component of the ubiquitin ligase SCF complex, in the circadian clock mechanism SLIMB (a homolog of β-Trcp) is a protein essential for rhythmic oscillations of Drosophila PER and a functioning clock in Drosophila. Considering the extensive homology between Drosophila and mammalian systems, it is of particular interest to study if a mammalian homologue of slimb, β-Trcp1, plays any role in the mammalian clockwork. Our preliminary results suggest that β-Trcp1 may participate in the mammalian clock mechanism by regulating mPER turnover. To confirm this result and unravel the details of how β-Trcp1 regulates the mammalian clockwork, we are characterizing molecular and behavioral circadian rhythms of β-Trcp1 knockout mice. Isolation of novel mPER-interacting components by affinity purification Based on our previous studies, the in vivo size of mPER-containing complexes rages up to 2.5 MDa. If a complex contains each of the 9 known clock proteins, it would weigh only 0.8 MDa. Therefore, the size of the complexes implies the presence of as yet unidentified components. We hypothesize that at least, some of components are involved in the posttranslational regulation of mPER proteins. These complexes will be purified by affinity purification using antibodies raised against clock proteins such as mCRY and mPER. The antibodies we generated have been thoroughly characterized and highly efficient for this purpose. Indeed, in a control experiment, we could obtain BMAL1 by affinity purification using anti-CLOCK antibody. The identity of copurified BMAL1 was confirmed by mass spec analysis. Once we have novel candidate proteins, we will generate antibodies against these proteins to confirm the interaction with known clock proteins, and study circadian regulation of the proteins.
Current Laboratory Members
Suhwan Chang, Ph.D KAIST, Korea, ‘03 Postdoctoral fellow Dillon Fritz, B.S. FSU, ‘02 Graduate Student, College of Medicine
Selected References
Lee, H., Chen, R., Lee, Y., Yoo, S. and Lee, C. Essential roles of CKId and CKIe in the mammalian circadian clock. Proceedings of the National Academy of Sciences (PNAS) in press   Chen, R., Schirmer, A., Lee, Y., Lee, H., Kumar, V., Yoo, S., Takahashi, J.S. and Lee, C. Rhythmic mPER abundance defines a critical nodal point for negative feedback within the circadian clock mechanism. Molecular Cell in press   Ramsey, K.M., Yoshino, J., Brace, C.S., Abrassart, D., Kobayashi, Y., Marcheva, B., Hong, H., Chong, J.L., Buhr, E.D., Lee, C., Takahashi, J.S. and Imai, S., Bass, J. (2009) Circadian Clock Feedback Cycle Through NAMPT-Mediated NAD+ Biosynthesis. Science 324:651.   Ansari, N., Agathagelidis, M.,Lee, C, Korf, H. and von Gall, C. (2009) Differential maturation of circadian rhythms in clock gene proteins in the suprachiasmatic nucleus and the pars tuberalis during mouse ontogeny. European Journal of Neuroscience. 29:477-89   Chen, R., Seo, D., Bell, E., von Gall, C. and Lee, C. (2008) Strong resetting of the mammalian clock by constant light followed by constant darkness. Journal of Neuroscience. 28:1839-47   Antoch, M.P., Gorbacheva, V.Y., Vykhovanets, O., Toshkov, I.A., Kondratov, R.V., Kondratova, A.A., Lee, C. and Nikitin, A.Y. (2008) Disruption of the circadian clock due to the Clock mutation has discrete effects on aging and carcinogenesis. Cell Cycle 7:1197-204.   Siepka, S. M., Yoo, S., Park, J., Lee, C. and Takahashi, J.S. (2007) Genetics and Neurobiology of Circadian Clocks in Mammals. Cold Spring Harbor Symposia on Quantitative Biology 72:251-9.   Busino L, Bassermann F, Maiolica A, Lee C, Nolan P.M., Godinho S, Draetta G.F. and Pagano., M. (2007) SCFFbxl3 Controls the Oscillation of the Circadian Clock by Directing the Degradation of Cryptochrome Proteins. Science 316:900-4.   Siepka S.M., Yoo S, Park J, Song W, Kumar V, Hu Y, Lee C and Takahashi J.S. (2007) Circadian Mutant Overtime Reveals F-box Protein FBXL3 Regulation of Cryptochrome and Period Gene Expression. Cell 129:1011-23.   Kondratov RV, Kondratova AA, Lee C, Gorbacheva VY, Chernov MV, Antoch MP. (2006) Post-Translational Regulation of Circadian Transcriptional CLOCK(NPAS2)/BMAL1 Complex by CRYPTOCHROMES. Cell Cycle 5: 890-5.   Yoo, S.H., Ko, C., Lowrey, P., Buhr, E., Song, E.J., Chang, S., Yoo, O.J., Yamazaki, S., Lee, C. and Takahashi, J.S. (2005) A novel E-box Enhancer Drives Mouse Period2 Circadian Oscillations in vivo. PNAS 102: 2608-2613   Lee, C., Weaver, D.R., and Reppert, S.M. (2004) Direct association between mouse PERIOD and CKIe is critical for a functioning circadian clock. Molecular and Cellular Biology.24: 584-594 Current Grant Support PI: Choogon Lee 5/18/2006-2/28/2011 NIH/NINDS, R01-NS-053616 $1,619,678 Roles of Casein Kinase Ie/d and b-Trcp in the mammalian circadian clock.