My lab is currently carrying out four research projects aimed at understanding how circadian clocks keep time, where and when they start to operate, and how they control overt rhythms.
1. How do circadian clocks maintain a period of ~24 hours?
The circadian timekeeping mechanism in fungi, plants and animals is based on a core transcriptional feedback loop. In Drosophila, CLOCK (CLK) and CYCLE (CYC) transcription factors form a heterodimer and bind to E-box regulatory elements during mid-day to activate expression of the period (per) and timeless (tim) genes. per and tim mRNAs accumulate to high levels early at night, but high levels of PER and TIM proteins don’t accumulate until late night due to phosphorylation by several protein kinases. PER and TIM then bind CLK-CYC and repress their own (and other) genes’ transcription until they are destroyed around mid-day, thereby triggering the next cycle of CLK-CYC transcriptional activation. The key components of this feedback loop are well conserved among different animal species.
The various biochemical steps that comprise the core feedback loop, when added together, should take much less than 24 hours to complete, which implies that delays are built into the core loop. PER, TIM and CLK proteins are phosphorylated coincident with transcriptional repression, and PER is required for CLK phosphorylation.
To understand how delays are incorporated into the core loop, we are determining how PER, TIM and CLK phosphorylation is controlled. Genetic and molecular approaches are being taken to identify CLK phosphorylation sites, identify the kinases and phosphatases that regulate PER, TIM and CLK phosphorylation, and determine the function of phosphorylating PER, TIM and CLK as specific sites.
2. Do interlocked feedback loops contribute to circadian timekeeping and output?
The core feedback loop is interlocked with the Clk feedback loop. In this loop, CLK-CYC activates transcription of vrille (vri) and Pdp1 epsilon/delta (Pdp1e/d). VRI protein accumulates to peak levels during the early night coincident with vri mRNA and binds VRI/PDP1e/d (V/P) elements to inhibit Clk transcription. PDP1e/d accumulates to peak levels as VRI levels decline during late night and binds V/P elements to promote Clk transcription (and transcription of other output genes) along with an independent constitutive Clk activator. Thus Clk transcripts accumulate in the opposite time of day compared to per, tim, vri and Pdp1e/d. Once CLK-CYC mediated transcription is inhibited by PER and TIM, vri and Pdp1e/d transcription declines until PER and TIM degradation initiates the next cycle of CLK-CYC transcription. A second interlocked loop also involves the clockwork orange (cwo) gene, which is activated by CLK-CYC, and then CWO protein is thought to feed back late at night to inhibit transcription by competing with CLK-CYC for E-box binding.
Since vri null mutants are lethal, it hasn’t been possible to determine whether Clk mRNA cycling is necessary for circadian timekeeping. A cwo null mutant decreases CLK-CYC dependent transcription and lengthens circadian period, which conflicts with the transcriptional repression function attributed to CWO based on in vitro and cell culture studies.
To determine whether the Clk loop contributes to circadian timekeeping, we are using transgenes designed to inducibly inactivate vri. We are also developing molecular and genetic tools to determine how cwo functions within the circadian timekeeping mechanism.
3. How are circadian oscillator cells determined during development?
The core and interlocked feedback loops are initiated by CLK-CYC mediated transcription. Circadian oscillators in dorsal and later brain neurons control locomotor activity rhythms in adults, and are known to operate as early as the first larval instar. We developed an antibody to CLK protein and showed that it is expressed in these presumptive brain oscillator neurons late during embryogenesis. Although PER is expressed independent of CLK in early embryos, PER accumulation in CLK expressing brain neurons is delayed ~6-8 hours, which implies that circadian oscillator function commences as soon as CLK is expressed in these neurons. In contrast to brain oscillator neurons, oscillator cells in adult peripheral tissues do not begin to operate until late in metamorphosis. Since CYC is required for oscillator function, we expect that cyc will be expressed in oscillator cells along with Clk, but no reagents for detecting CYC are available to test this possibility. CYC is also required for processes other than circadian clocks, most notably the homeostatic control of sleep, but whether CYC functions in oscillator cells to regulate sleep homeostasis is not known.
To understand how oscillator cells are determined during development, we are using genetic screens and tissue-specific Clk transcriptional regulatory elements to identify Clk transcriptional activators. We have used recombineering technology to produce cyc transgenes that express epitope tagged CYC protein, which enables detection in fly tissues, and will therefore be used to determine whether CYC is co-expressed with CLK in oscillator cells and whether CYC expression in oscillator cells controls sleep homeostasis.
4. How are rhythms in olfactory and gustatory physiology and behavior regulated?
Circadian clocks are found in the brain and many peripheral tissues throughout the fly. However, little is known about the rhythms controlled by different oscillator tissues, with the exception of locomotor activity rhythms, which are controlled by dorsal and lateral brain neurons. We discovered that clocks in olfactory sensory neurons, which mainly reside in the antenna, control rhythms in the sensitivity to odors that peak in the middle of the night. Although these rhythms in sensitivity were identified by measuring odor-induced electrophysiological responses, rhythms in the amplitude of spontaneous activity from olfactory sensory neurons implies that their basic membrane physiology is under clock control. The clock imparts rhythms in olfactory sensory neuron physiology by controlling the levels of G-protein coupled receptor kinase 2, which in turn drives rhythms in the accumulation of odorant receptors in olfactory sensory neuron dendrites, which is where odors are detected. These physiological rhythms control behavioral rhythms in attraction and repulsion to various odors. We recently found that rhythms in the detection of chemical compounds extend to the gustatory system, where sensitivity to bitter and sweet flavors peak early in the morning. Like olfactory rhythms, gustatory rhythms are also controlled by G-protein coupled receptor kinase 2, but this kinase represses gustatory responses and promotes olfactory responses, consistent with the opposite peaks in sensitivity of these rhythms. Clocks in gustatory sensory neurons control behavioral rhythms in the extension of the proboscis (the fly tongue equivalent) to investigate tastants, and suppress feeding.
To understand how rhythms in olfaction are regulated, we are using biochemical, molecular and genetic approaches to determine how G-protein coupled receptor kinase 2 promotes odorant receptor accumulation in dendrites, how rhythms in the amplitude of olfactory sensory neuron activity are propagated through the neuronal circuit, how G-protein coupled receptor kinase 2 promotes olfactory responses and represses gustatory responses, and how rhythms in chemosensory neuron physiology control rhythms in chemosensory behavior.
