My lab is currently carrying out four research projects aimed at understanding how the core and interlocked feedback loops keep time, how circadian clocks develop and initiate function, and how circadian clocks control physiological and behavioral 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 Clktranscriptional 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 physiological and behavioral rhythms 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 brain pacemaker neurons. We previously discovered that clocks in olfactory and gustatory sensory neurons, which mainly reside in the antenna and the proboscis, respectively, control rhythms in the sensitivity to chemicals in the environment. Our current work focuses on the regulation of locomotor activity rhythms via daily changes in pacemaker neuron arborization and the accumulation of PIGMENT DISPERSING FACTOR (PDF) neuropeptide. We recently discovered that the interlocked feedback loop repressor VRI is necessary for rhythms in pacemaker neuron arborization and PDF accumulation. Since VRI controls transcription that peaks near dawn, VRI is thought to regulate key genes that control neuronal plasticity and PDF expression.
Rhythms in pacemaker neuron arborization and PDF accumulation are known to be altered by the transcription factor MEF2, the GTP binding protein PURA, the RHO1 GTPase, and the metalloprotease MMP1. Our current experiments are aimed at determining how VRI integrates with these other regulators to understand the molecular mechanisms that underly pacemaker neuron arborization and PDF accumulation. Such work will provide insight into the clock regulation of a primary behavioral parameter: locomotor activity rhythms.