A novel C-terminal domain of Drosophila PERIOD inhibits dCLOCK:CYCLE-mediated transcription

The essence of the Drosophila circadian clock involves an autoregulatory feedback loop in which PERIOD (PER) and TIMELESS (TIM) inhibit their own transcription by association with the transcriptional activators dCLOCK (dCLK) and CYCLE (CYC) [1]. Because PER, dCLK, and CYC each contain a PAS domain [1], it has been assumed that these interaction domains are important for negative feedback. However, a critical role for PAS-PAS interactions in Drosophila clock function has not been shown. Nuclear transport of PER is also believed to be an essential regulatory step for negative feedback [1-3], but this has not been directly tested, and the relevant nuclear localization sequence (NLS) has not been functionally mapped. We evaluated these critical aspects of PER-mediated transcriptional inhibition in Drosophila Schneider 2 (S2) cells. We mapped the dCLK:CYC inhibition domain (CCID) of PER and discovered that it lies in the C terminus, downstream of the PAS domain. Using deletion mutants and site-directed mutagenesis, we identified a novel NLS in the CCID of PER that is a potent regulator of PER's nuclear transport in S2 cells. We further found that nuclear transport, primarily through this novel NLS, is essential for the inhibitory activity of PER. The data indicate that nuclear PER inhibits dCLK:CYC-mediated transcription through a novel domain that additionally contains a potent NLS.