| Research Abstract |
In the eukaryotic circadian model systems, translocation of the oscillatory gene products into the nucleus is a key step for generation of a 24 hour cycle of the biological clock. We have examined nuclear import of clock proteins of the mammalian period gene family and the effect of serum shock, which induces a synchronous clock in cultured cells. We examined the nuclear import of mPER proteins in COS7 cells and found that nuclear translocation of mPER1 and mPER2 involves physical interactions with mPER3. This indicates that nuclear translocation of mPER1 also can occur under physiological conditions in the absence of CRY proteins.
Recently we demonstrated that an accessory transcription loop exists helping to the core clock feedback loop. Transcript levels of DBP, a member of the PAR leucine zipper transcription factor family, exhibit a robust rhythm in the SCN.We report that DBP is able to activate the promoter of a putative clock oscillating gene, mPer1, by directly binding to the mPer1 promoter. DBP and CLOCK-BMAL1 cooperatively activate the mPer1 promoter. On the other hand, dbp transcription is activated by CLOCK-BMAL1 through E-boxes and inhibited by the mPER and mCRY proteins, as is the case for mPer1. Antiphase circadian regulated E4BP4, another member of leucine zipper transcription factor without PAR domain, antagonistically suppresses mPer1 transcription. Thus, a clock-controlled dbp and e4bp4 genes may play an important role in the central clock oscillation.
Using transgenic mice carrying the mPer1 promoter fused to the luciferase (mPer1-luc) gene, we recently succeeded to monitor luciferase-mediated bioluminescence with a day-night variation in the SCN in brain slices and in living animals. We can record for several days oscillating photon emission with a periodicity and phase that accurately mirrored native mPer1 mRNA expression. The real-time optical imaging of gene expression will be a new powerful tool to study mammalian brain function.
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