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The core oscillator that generates circadian rhythm in eukaryotes consists of transcription/translation-based autoregulatory feedback loops by which clock gene products negatively regulate their own expression. Control of the accumulation and nuclear entry of the negative regulators PER and CRY is believed to be a key step in these loops. We clarified the mutual interaction between zebrafish clock-related proteins and their sub-cellular localizations in NIH3T3 cells. Six CRYs exist in zebrafish, of which zCRY1a strongly represses zCLOCK1: zBMAL3-mediated transcription, but zCRY3 does not. We show that zCRY1a interacts with zCLOCK1 and zBMAL3, facilitating nuclear accumulation, whereas zCRY3 associates with neither one and does not influence their sub-cellular distributions. We cloned zPer2 cDNA and showed that the protein product encoded by the cDNA acts as a moderate transcriptional repressor. In our sub-cellular localization studies we also found that zPER2 interacts with the zCLOCK1:zBMAL3 heterodimer, causing its cytoplasmic retention. zCRY1a and zPER2 apparently have opposite effects on the sub-cellular distribution of zCLOCK:zBMAL heterodimer. We speculate that the opposite regulation of the sub-cellular distribution of this is associated with the different transcriptional repression abilities of zCRY1a and zPER2. zCRY1a acts as a potent transcriptional inhibitor by interacting directly with the zCLOCK:zBMAL heterodimer in the nucleus, whereas zPER2 maintains the zCLOCK:zBMAL heterodimer in the cytoplasm, resulting in transactivation repression.

New role of zCRY and zPER2 as regulators of sub-cellular distributions of zCLOCK and zBMAL proteins