A Role for the PERIOD:PERIOD Homodimer in the Drosophila Circadian Clock

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Abstract
Circadian clocks in eukaryotes rely on transcriptional feedback loops, in which clock genes repress their own transcription resulting in molecular oscillations with a period of ∼24 h. In Drosophila, the clock proteins Period (PER) and Timeless (TIM) operate in such a feedback loop, whereby they first accumulate in the cytoplasm of clock cells as a heterodimer. Nuclear translocation of the complex or the individual PER and TIM proteins is followed by repression of per and tim transcription, whereby PER seems to act as the prime repressor. We found that in addition to PER:TIM complexes, functional PER:PER homodimers exist in flies. Specific disruption of PER homodimers results in drastically impaired behavioral and molecular rhythmicity, pointing the biological importance of this clock protein complex. Analysis of PER subcellular distribution and repressor competence in the PER dimer mutant revealed defects in PER nuclear translocation and a disruption of rhythmic period transcription. The striking similarity of these phenotypes with that of reduced CKII activity suggests that the formation or function of the PER dimer is closely linked to this kinase. Our results confirm a previous structural model for PER and provide strong evidence that PER homodimers are important for circadian clock function. The current models of circadian clocks in flies and mammals involve the formation of complexes between clock proteins in the cytoplasm. These complexes are usually heterodimers (that is, made up of two different clock proteins) and appear to enter the nucleus at certain times of the circadian day in order to shut down their own gene expression by deactivating specific transcription factors. After progressive phosphorylation the repressor proteins eventually are degraded so that a new cycle of transcription can begin. Here we present evidence that in addition to heterodimeric complexes, the clock protein PERIOD (PER) also forms homodimers (pairs of identical proteins). Based on a structural model a PER mutant was designed, which is not able to form homodimers but can still bind to its partner TIMELESS (TIM). Flies expressing this mutant PER protein show abnormal clock function in regard to PER nuclear translocation, repressor activity, and behavioral rhythms. The circadian clock model in flies therefore needs to be extended by adding the PER:PER homodimer as a functional unit. Recent structural studies with mammalian PER proteins suggest that homodimers between clock proteins are an important general feature of eukaryotic clocks.

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