The Drosophila circadian clock consists of two interlocked transcriptional feedback loops. In one loop, dCLOCK/CYCLE activates period expression, and PERIOD protein then inhibits dCLOCK/CYCLE activity. dClock is also rhythmically transcribed, but its regulators are unknown. vrille (vri) and Par Domain Protein 1 (Pdp1) encode related transcription factors whose expression is directly activated by dCLOCK/CYCLE. We show here that VRI and PDP1 proteins feed back and directly regulate dClock expression. Repression of dClock by VRI is separated from activation by PDP1 since VRI levels peak 3-6 hours before PDP1. Rhythmic vri transcription is required for molecular rhythms, and here we show that the clock stops in a Pdp1 null mutant, identifying Pdp1 as an essential clock gene. Thus, VRI and PDP1, together with dClock itself, comprise a second feedback loop in the Drosophila clock that gives rhythmic expression of dClock, and probably of other genes, to generate accurate circadian rhythms.

The Period (PER), Timeless (TIM), and Double-Time (DBT) proteins are essential components of one feedback loop in the Drosophila circadian molecular clock. PER and TIM physically interact. Coexpression of PER and TIM promotes their nuclear accumulation and influences the activity of DBT: although DBT phosphorylates and destabilizes PER, this is suppressed by TIM. Experiments using Drosophila cells in culture have indicated that PER can translocate to the nucleus without TIM and will repress transcription in a DBT-potentiated manner. In this study, we examined the control of PER subcellular localization in Drosophila clock cells in vivo. We found that PER can translocate to the nucleus in tim 01 null mutants but only if DBT kinase activity is inhibited. We also found that nuclear PER is a potent transcriptional repressor in dbt mutants in vivo without TIM. Thus, in vivo, DBT regulates PER subcellular localization, in addition to its previously documented role as a mediator of PER stability. However, DBT does not seem essential for transcriptional repression by PER. It was reported previously that overexpression of a second kinase, Shaggy (SGG)/Glycogen Synthase Kinase 3, accelerates PER nuclear accumulation. Here, we show that these effects of SGG on PER nuclear accumulation require TIM. We propose a revised clock model that incorporates this tight kinase regulation of PER and TIM nuclear entry.Key words: circadian; Period; Double-Time; Timeless; nuclear entry; Drosophila IntroductionRhythms of gene expression and protein levels lie at the core of the molecular clocks that regulate circadian (ϳ24 h) rhythms of behavior and physiology. The best-studied circadian feedback loop involves the Drosophila clock genes period (per) and timeless (tim). Many components of this negative feedback loop play similar roles in the mammalian clock (Panda et al., 2002;Stanewsky, 2003).In Drosophila, per and tim transcription is activated by Clock (CLK)/Cycle (CYC) heterodimers binding to E-boxes in the per and tim promoters. PER and TIM proteins heterodimerize in the cytoplasm, and, until recently (see below), it was believed that they enter the nucleus together. PER is more stable than TIM in the nucleus and represses additional per and tim transcription by directly inhibiting CLK/CYC.The PER/TIM loop is characterized by delays that separate transcription from repression, important for rhythmic, as opposed to continuous, negative feedback. Peak per RNA and protein levels are separated by ϳ6 h at least partly by Double-Time (DBT)/Casein Kinase 1⑀ (CK1⑀), a protein kinase that destabilizes cytoplasmic PER while TIM levels are low Suri et al., 2000). Thus, PER levels are constitutively low in tim 01 mutants (Price et al., 1995). DBT stably associates with PER (Kloss et al., 2001) and phosphorylates PER Ko et al., 2002) (S. Kivimae and M. W. Young, unpublished data), which leads to PER ubiquitination and degradation (Grima et al., 2002;Ko et al., 2002). These in vivo findings led to the model that rising TIM levels allow the formation...