|Subject||Division of Physiology and Systems Bioscience|
Lecturer Hitoshi Inokawa, PhD.Lecturer Nobuya Koike, PhD.
Lecturer Yoshiki Tsuchiya, PhD.
Assistant Professor Yoichi Minami, PhD.
Assistant Professor Yasuhiro Umemura, PhD.
In mammals, various physiological aspects (such as endocrine function, energy metabolism, and behavior) show a near 24-hour rhythm that is controlled by an internal circadian clock system. Although the central pacemaker resides in the suprachiasmatic nucleus, most peripheral cells also have their own circadian clock. We have shown that even in cultured cell lines, each cell contains its own intrinsic circadian oscillator (Yagita et al, Science, 2001). Based on these findings, we have been investigating the mammalian circadian clock system using cell lines as a model tool of the molecular clock.
Fibroblast cell lines as model system of mammalian circadian clock
Using fibroblast cell lines such as NIH3T3 and rat-1 cells, we have investigated the cellular circadian clock system. The circadian clock system is likely to consist of not only a transcriptional feedback loop-based core oscillator but also complex networks including various levels of regulation. To dissect the circadian clock system, we chose a few approaches to understand the mammalian circadian clock system at the cellular level.
Identification of important functional domains of clock proteins for circadian clock oscillation in living cells.
We succeeded in developing high throughput real-time monitoring of cellular circadian oscillation using the mPer2 promoter and the mBMAL1 promoter-driven luciferase reporter. Using this system, we analyzed mutant proteins of mBMAL1 clock protein generated through random mutagenesis. To assay the circadian oscillation of bioluminescence expressing these mutants, we identified critical mutants that impair the endogenous circadian clock system. After detailed analysis of the molecular mechanism of this mutant, we have shown that the C-terminal domain of BMAL1 plays a key role in eliciting the “cyclic transcriptional regulation” in mammalian cells (Kiyohara et al, PNAS, 2006).
Protein Dynamics of Circadian Clock
Not only the transcriptional dynamics but also the dynamics of clock proteins including posttranslational modification is important for the mammalian circadian clock system. We investigated regulation mechanisms of subcellular localization of clock proteins such as mPER and mCRY (Yagita et al, Genes Dev., 2000) (Yagita et al, EMBO J., 2002), and protein dynamics of the mPER2 protein (Yamamoto et al., Mol. Cell. Biol., 2005) (Nishii et al, Neurosci Lett, 2006). These studies have revealed that posttranscriptional/posttranslational regulation mechanisms play distinct roles to the mammalian circadian clock system.
Development of cellular circadian clock
We have studied the mechanisms of circadian clock development. Recent studies reported that, core circadian genes have been found to express in mouse fertilizing eggs and preimplantation embryos, however, they did not show circadian periodicity of those clock gene expression. In contrast with the evidences that most of somatic cells in our body have their own circadian clock oscillator, it is suggested that the circadian clock is likely to develop during the embryonic period. Therefore we established in vitro assay system to investigate the development of circadian clock oscillation during the cellular differentiation from ES cells in vitro.
Prevously, we show that the circadian bioluminescence activity rhythm is not detected in the mouse embryonic stem (ES) cells. We also show that the apparent circadian clock oscillation is induced during the differentiation culture of mouse ES cells without maternal entraining factors. In addition, when those differentiated cells are reprogrammed by expressing Sox2, Klf4, Oct-3/4 and c-Myc genes, those are used to generate induced pluripotent stem (iPS) cells, the circadian oscillation re-disappeared (Yagita et al, PNAS, 2010). These results demonstrate that the intrinsic program controls the circadian oscillator formation during the differentiation process of ES cells in cultural condition.