Michael W. Young

double-time is a novel Drosophila clock gene that regulates PERIOD protein accumulation

We have isolated three alleles of a novel Drosophila clock gene, double-time (dbt). Short- (dbtS) and long-period (dbtL) mutants alter both behavioral rhythmicity and molecular oscillations from previously identified clock genes, period and timeless. A third allele, dbtP, causes pupal lethality and eliminates circadian cycling of per and tim gene products in larvae. In dbtP mutants, PER proteins constitutively accumulate, remain hypophosphorylated, and no longer depend on TIM proteins for their accumulation. We propose that the normal function of DOUBLETIME protein is to reduce the stability and thus the level of accumulation of monomeric PER proteins. This would promote a delay between per/tim transcription and PER/TIM complex function, which is essential for molecular rhythmicity.

The Drosophila Clock Gene double-time Encodes a Protein Closely Related to Human Casein Kinase Iε

The cloning of double-time (dbt) is reported. DOUBLETIME protein (DBT) is most closely related to human casein kinase Iepsilon. dbtS and dbtL mutations, which alter period length of Drosophila circadian rhythms, produce single amino acid changes in conserved regions of the predicted kinase. dbtP mutants, which eliminate rhythms of per and tim expression and constitutively overproduce hypophosphorylated PER proteins, abolish most dbt expression. dbt mRNA appears to be expressed in the same cell types as are per and tim and shows no evident oscillation in wild-type heads. DBT is capable of binding to PER in vitro and in Drosophila cells, suggesting that a physical association of PER and DBT regulates PER phosphorylation and accumulation in vivo.

Light-Induced Degradation of TIMELESS and Entrainment of the Drosophila Circadian Clock

Two genes, period (per) and timeless (tim), are required for production of circadian rhythms in Drosophila. The proteins encoded by these genes (PER and TIM) physically interact, and the timing of their association and nuclear localization is believed to promote cycles of per and tim transcription through an autoregulatory feedback loop. Here it is shown that TIM protein may also couple this molecular pacemaker to the environment, because TIM is rapidly degraded after exposure to light. TIM accumulated rhythmically in nuclei of eyes and in pacemaker cells of the brain. The phase of these rhythms was differentially advanced or delayed by light pulses delivered at different times of day, corresponding with phase shifts induced in the behavioral rhythms.

vrille, Pdp1, and dClock Form a Second Feedback Loop in the Drosophila Circadian Clock

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.

Cycling vrille Expression Is Required for a Functional Drosophila Clock

We identified a novel regulatory loop within Drosophilas circadian clock. A screen for clock-controlled genes recovered vrille (vri), a transcription factor essential for embryonic development. vri is expressed in circadian pacemaker cells in larval and adult brains. vri RNA levels oscillate with a circadian rhythm. Cycling is directly regulated by the transcription factors dCLOCK and CYCLE, which are also required for oscillations of period and timeless RNA. Eliminating the normal vri cycle suppresses period and timeless expression and causes long-period behavioral rhythms and arrhythmicity, indicating that cycling vri is required for a functional Drosophila clock. We also show that dCLOCK and VRI independently regulate levels of a neuropeptide, pigment dispersing factor, which appears to regulate overt behavior.

Isolation of timeless by PER Protein Interaction: Defective Interaction Between timeless Protein and Long-Period Mutant PERL

The period (per) gene likely encodes a component of the Drosophila circadian clock. Circadian oscillations in the abundance of per messenger RNA and per protein (PER) are thought to arise from negative feedback control of per gene transcription by PER. A recently identified second clock locus, timeless (tim), apparently regulates entry of PER into the nucleus. Reported here are the cloning of complementary DNAs derived from the tim gene in a two-hybrid screen for PER-interacting proteins and the demonstration of a physical interaction between the tim protein (TIM) and PER in vitro. A restricted segment of TIM binds directly to a part of the PER dimerization domain PAS. PERL, a mutation that causes a temperature-sensitive lengthening of circadian period and a temperature-sensitive delay in PER nuclear entry, exhibits a temperature-sensitive defect in binding to TIM. These results suggest that the interaction between TIM and PER determines the timing of PER nuclear entry and therefore the duration of part of the circadian cycle.

Mammalian Circadian Autoregulatory Loop: A Timeless Ortholog and mPer1 Interact and Negatively Regulate CLOCK-BMAL1-Induced Transcription

We report the cloning and mapping of mouse (mTim) and human (hTIM) orthologs of the Drosophila timeless (dtim) gene. The mammalian Tim genes are widely expressed in a variety of tissues; however, unlike Drosophila, mTim mRNA levels do not oscillate in the suprachiasmatic nucleus (SCN) or retina. Importantly, hTIM interacts with the Drosophila PERIOD (dPER) protein as well as the mouse PER1 and PER2 proteins in vitro. In Drosophila (S2) cells, hTIM and dPER interact and translocate into the nucleus. Finally, hTIM and mPER1 specifically inhibit CLOCK-BMAL1-induced transactivation of the mPer1 promoter. Taken together, these results demonstrate that mTim and hTIM are mammalian orthologs of timeless and provide a framework for a basic circadian autoregulatory loop in mammals.

Rhythmic expression of timeless: A basis for promoting circadian cycles in period gene autoregulation

The clock gene timeless (tim) is required for circadian rhythmicity in Drosophila. The accumulation of tim RNA followed a circadian rhythm, and the phase and period of the tim RNA rhythm were indistinguishable from those that have been reported for per. The tim RNA oscillations were found to be dependent on the presence of PER and TIM proteins, which demonstrates feedback control of tim by a mechanism previously shown to regulate per expression. The cyclic expression of tim appears to dictate the timing of PER protein accumulation and nuclear localization, suggesting that tim promotes circadian rhythms of per and tim transcription by restricting per RNA and PER protein accumulation to separate times of day.

Regulation of nuclear entry of the Drosophila clock proteins period and timeless.

Two genes, period (per) and timeless (tim), are essential for circadian rhythmicity in Drosophila. The encoded proteins (PER and TIM) physically interact. Here, it is shown that TIM and PER accumulate in the cytoplasm when independently expressed in cultured (S2) Drosophila cells. However, the proteins move to the nuclei of these cells if coexpressed. Domains of PER and TIM have been identified that block nuclear localization of the monomeric proteins. In vitro protein interaction studies indicate that the sequence inhibiting the nuclear accumulation of PER forms a binding site for TIM. The results indicate a mechanism for controlled nuclear localization in which suppression of cytoplasmic localization is accomplished by direct interaction of PER and TIM. No other clock functions are required for nuclear localization. The findings suggest that a checkpoint in the circadian cycle is established by requiring cytoplasmic assembly of a PER/TIM complex as a condition for nuclear transport of either protein.

Positional Cloning and Sequence Analysis of the Drosophila Clock Gene, timeless

The Drosophila genes timeless (tim) and period (per) interact, and both are required for production of circadian rhythms. Here the positional cloning and sequencing of tim are reported. The tim gene encodes a previously uncharacterized protein of 1389 amino acids, and possibly another protein of 1122 amino acids. The arrhythmic mutation tim01 is a 64-base pair deletion that truncates TIM to 749 amino acids. Absence of sequence similarity to the PER dimerization motif (PAS) indicates that direct interaction between PER and TIM would require a heterotypic protein association.