The main goal of our laboratory is to understand the molecular and biochemical bases of biological clocks. To achieve this goal. we are using the powerful genetics available in Drosophila in combination with biochemical. molecular and histochemical approaches.
Daily fluctuations in biochemical. physiological and behavioral phenomena are governed by endogenous circadian (~24 hour) clocks that can be synchronized (entrained) by external time cues (zeitgebers). most notably the daily changes in light/dark and temperature. This adaptive feature of circadian clocks enables organisms to temporally align their physiology and behavior such that they occur at biologically advantageous times during the day.
The isolation of "clock genes" has provided significant insights into the molecular underpinnings governing circadian rhythms. A common theme in clocks from bacteria to humans is that at the "heart" of these pacemakers lie transcriptional-translational feedback loops. The best characterized animal model system for a circadian clock is Drosophila melanogaster. where four clock proteins termed PERIOD (PER). TIMELESS (TIM). dCLOCK and CYCLE (CYC) function in a negative transcriptional autoregulatory loop. dCLOCK and CYC are members of the basic-helix-loop-helix (bHLH)/PAS (PER-ARNT-SIM) superfamily of transcription factors and are required for the daily stimulation of per and tim expression. PER and TIM form a complex in the cytoplasm that enters the nucleus in a temporally gated manner where they bind the dCLOCK-CYC heterodimer blocking its DNA binding activity. In the absence of denovo synthesis. the concentrations of PER and TIM in the nucleus decrease below threshold levels relieving autoinhibition. enabling the next round of per and tim transcript accumulation. Posttranscriptional mechanisms play an important role because they introduce "biochemical time constraints" that stretch the transcriptional feedback loop to ~24 hr and also allows it to respond to external stimuli. For example. light evokes the rapid degradation of TIM. the primary clock-specific photoresponse resetting the oscillatory mechanism. A blue-light photoreceptor called CRYPTOCHROME (CRY) has been implicated in transducing photic signals to TIM. Furthermore. the cytoplasmic phosphorylation of PER by the kinase DOUBLE-TIME (DBT) renders PER unstable. Cytoplasmic PER is stabilized by interacting with TIM which ensures that the accumulation and nuclear entry of the PER-TIM complex is a slow process creating a time-window for daily increases in the levels of per and tim transcripts. Our studies are geared towards isolating all the components that comprise a circadian timekeeping device and understanding how the daily changes in visible light and ambient temperature modulate the oscillatory mechanism.