Division of Physiology and Systems Bioscience

Subject Division of Physiology and Systems Bioscience
 Kazuhiro Yagita, MD. PhD
Lecturer Hitoshi Inokawa, PhD.
Lecturer Nobuya Koike, PhD.
Lecturer Yoshiki Tsuchiya, PhD.

Assistant Professor Yoichi Minami, PhD.

Assistant Professor Yasuhiro Umemura, PhD.

Research Contents

  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.
  1. Kunimoto T, Okubo N, Minami Y, Fujiwara H, Hosokawa T, Asada M, Oda R, Kubo T, Yagita K*. A PTH-responsive circadian clock operates in ex vivo mouse femur fracture healing site. Sci Rep., 29, 6:22409,  2016 (*Corresponding author)
  2. Tsuchiya Y, Umemura Y, Minami Y, Koike N, Hosokawa T, Hara M, Ito H, Inokawa H, Yagita K*. Effect of Multiple Clock Gene Ablations on the Circadian Period Length and Temperature Compensation in Mammalian Cells. J Biol Rhythms. 31, 48-56, 2016  (*Corresponding author)
  3. Hosokawa T, Tsuchiya Y, Okubo N, Kunimoto T, Minami Y, Fujiwara H, Umemura Y, Koike N, Kubo T, Yagita K*. Robust Circadian Rhythm and Parathyroid Hormone-Induced Resetting during Hypertrophic Differentiation in ATDC5 Chondroprogenitor Cells. Acta Histochem Cytochem. 48, 165-71, 2015.  (*Corresponding author)
  4. Tsuchiya Y, Minami Y, Umemura Y, Watanabe H, Ono D, Nakamura W, Takahashi T, Honma S, Kondoh G, Matsuishi T, Yagita K*. Disruption of MeCP2 attenuates circadian rhythm in CRISPR/Cas9-based Rett syndrome model mouse. Genes Cells, 20, 992-1005. 2015.  (*Corresponding author)
  5. Oshima T, Yamanaka I, Kumar A, Yamaguchi J, Nishiwaki-Okawa T, Muto K, Kawamura R, Hirota T, Yagita K, Irie S, Kay SA, Yoshimura T, Itami K., C-H Activation Generates Period-Shortening Moleculaes That Target Cryptochrome in the Mammalian Circadian Clock., Angew. Chem. Int. Ed., 54, 7193-7197, 2015
  6. Okubo N, Fujiwara H, Minami Y, Kunimoto T, Hosokawa T, Umemura Y, Inokawa H, Asada M, Oda R, Kubo T, Yagita K*., Parathyroid hormone resets the cartilage circadian clock of the organ-cultured murine femur., Acta Orthopedica, 86. 627-631. 2015.
  7. Umemura Y, Koike N, Matsumoto T, Yoo S-H, Zhen C, Yasuhara N, Takahashi JS, Yagita K*., Transcriptional Program of Kpna2 /Importin-2 Regulates Cellular Differentiation-Coupled Circadian Clock Development in Mammalian Cell, Proc. Natl. Acad. Sci. USA, 111, E5039-48, 2014 (*Corresponding author)
  8. Inada Y, Uchida H, Umemura Y, Nakamura W, Sakai T, Koike N, Yagita K*. Cell and Tissue-autonomous development of the circadian clock in mouse embryos. FEBS Lett., 588. 459-465, 2014. (*Corresponding author)
  9. Okubo N, Minami Y, Fujiwara H, Umemura Y, Tsuchiya Y, Shirai Y, Oda R, Inokawa H, Kubo T, Yagita K*. Prolonged Bioluminescence Monitoring in Mouse ex vivo Bone Culture Revealed Persistent Circadian Rhythms in Articular Cartilages and Growth Plates., PLoS One, 8, e78306, 2013 (*Corresponding author)
  10. Umemura Y, Yoshida J, Wada M, Tsuchiya Y, Minami Y, Watanabe H, Kondoh G, Takeda J, Inokawa H, Horie K, Yagita K*. An in vitro ES cell-based clock recapitulation assay model identifies CK2a as an endogenous clock regulator. PLoS One, 8, e67241, 2013 (*Corresponding author)
  11. Sumiyama K, Kawakami K, Yagita K. A Simple and highly efficient transgenesis method in mice with the Tol2 transposon system and cytoplasmic microinjection., Genomics, 95, 306-311, 2010.
  12. Yagita K*, Horie K, Koinuma S, Nakamura W, Yamanaka I, Urasaki A, Shigeyoshi Y, Kawakami K, Shimada S, Takeda J, Uchiyama Y, Development of circadian oscillator during differentiation of mouse embryonic stem cell in vitro., Proc. Natl. Acad. Sci. USA, 107, 3846-3851, 2010. (* Corresponding author)
  13. Yagita K*, Yamanaka I, Emoto N, Kawakami K, Shimada S, Real-time monitoring of circadian clock oscillations in primary culture of mammalian cells using Tol2 transposon-mediated gene transfer strategy. BMC Biotechnology, 10, 3, 2010. (*Corresponding author)
  14. Kiyohara, Y#, Nishii K#, Ukai-Tadenuma M, Ueda HR, Uchiyama Y, Yagita K*. Detection of a circadian enhancer in the mDbp promoter using prokaryotic transposon vector based strategy., Nucl. Acids Res., 36, e23, 2008. (*Corresponding Author)
  15. Kiyohara Y.B#, Tagao S#, Tamanini F, Morita A, Sugisawa Y, Yasuda M, Yamanaka I, Ueda H.R, van der Horst GTJ, Kondo T, and Yagita K*. The BMAL1 C terminus regulates the circadian transcription feedback loop. Proc. Natl. Acad. Sci. USA, 103, 10074-9, 2006. (*Corresponding Author)
  16. Yamaguchi S, Isejima H, Matsuo T, Okura R, Yagita K, Kobayashi M, Okamura H. Synchronization of cellular clocks in the suprachiasmatic nucleus. Science. ;302, 1408-12,2003
  17. Yagita K, Tamanini F, Yasuda M, Hoeijmakers JH, van der Horst GT, Okamura H. Nucleocytoplasmic shuttling and mCRY-dependent inhibition of ubiquitylation of the mPER2 clock protein. EMBO J., 21, 1301-1314: 2002
  18. Yagita K, Tamanini F, van Der Horst GT, Okamura H. Molecular mechanisms of the biological clock in cultured fibroblasts., Science., 292:278-81. 2001

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e-mail kyagita@koto.kpu-m.ac.jp