Chapter 5 - Molecular Genetic Analysis of Circadian Timekeeping in Drosophila
Introduction
Research on the fruit fly Drosophila melanogaster has had a profound impact on our understanding of the circadian timekeeping mechanism. Groundbreaking studies by Ron Konopka and Seymour Benzer identified the first “clock gene,” period (per), in a screen for mutants with altered free-running (in constant darkness or DD) circadian periods in the rhythm of adult emergence (Konopka and Benzer, 1971). As per mutants alter circadian period, per was thought to be integrally involved in keeping circadian time, but determining how per contributed to circadian timekeeping did not take off until the per gene was isolated at Brandeis University in the labs of Michael Rosbash and Jeff Hall and at Rockefeller University in Michael Young's lab (Bargiello and Young, 1984, Bargiello et al., 1984, Reddy et al., 1984, Zehring et al., 1984). The PER protein sequence did not offer many clues about its role in the clock because its only distinguishing features were a stretch of threonine–glycine repeats, later shown to be involved in adapting to different thermal environments (Sawyer et al., 1997), and a region similar to a portion of the Drosophila SINGLE-MINDED (SIM) and mammalian aryl hydrocarbon receptor nuclear translocator (Arnt) proteins, termed the Per–Arnt–Sim (PAS) domain (Nambu et al., 1991), that mediates protein–protein interactions (Huang et al., 1993). However, the discovery that per mRNA and protein cycle in a circadian manner (Hardin et al., 1990, Siwicki et al., 1988), and that PER protein is required for cycling of per mRNA (Hardin et al., 1990), suggested that per contributes to circadian timekeeping via a feedback loop in which PER protein controls rhythms in per mRNA expression (Hardin et al., 1990). Studies demonstrating transcriptional control of per mRNA cycling and PER-dependent inhibition of per mRNA expression further refined the role of PER in this feedback loop as a transcriptional repressor (Hardin et al., 1992, Zeng et al., 1994). Subsequent studies not only support the view that this transcriptional feedback loop keeps circadian time in Drosophila but also show that similar feedback loops keep circadian time in diverse eukaryotic species including plants, fungi, and animals, the latter of which even shares critical feedback loop components such as per (for reviews, see Bell-Pedersen et al., 2005, Dunlap, 1999, Young and Kay, 2001).
Although per is an essential feedback loop component, many other genes are required to sustain the per feedback loop. In Section II of this chapter, I explain the roles other genes play within the per feedback loop and describe how the per feedback loop relates to other interlocked feedback loops. As the per feedback loop has many components and is inextricably linked to other feedbacks loops, I refer to the per feedback loop as the “core loop” and to the combined core and interlocked feedback loops as “circadian feedback loops.” The different steps required to construct a transcriptional feedback loop (such as the core loop) can be completed in far less than ∼ 24 h, indicating that potent mechanisms have evolved to impart delays in feedback regulation. Section III focuses on how posttranscriptional regulation of key feedback loop components produces delays in transcriptional feedback that set the pace of the circadian oscillator. Entrainment of circadian feedback loops to environmental light–dark cycles is critical for driving physiological, metabolic, and behavioral rhythms at the appropriate time of day. Unlike the case in mammals, light can directly entrain circadian feedback loops in peripheral tissues from Drosophila, thus there is no intermediary “master” pacemaker in the Drosophila brain that relays light information to the periphery. However, light can entrain the network of Drosophila brain pacemaker neurons via multiple mechanisms depending on which cells detect the light. Section IV discusses how light entrains circadian feedback loops in different cells and tissues.
In Section V of this review, I summarize the main conclusions from each section, point out where there are gaps in our understanding, and discuss how filling these gaps may explain how these circadian feedback loops account for basic features of circadian clock such as the 24-h period and entrainment to environmental cycles. The general consensus is that circadian feedback loops sit at the heart of the circadian timekeeping mechanism in eukaryotes. However, experiments demonstrating that circadian rhythms in the phosphorylation/dephosphorylation of KaiC protein in cyanobacteria can be reconstituted in a test tube (Nakajima et al., 2005), and rhythms in the oxidation of peroxiredoxins in a primitive eukaryotic alga Ostreococcus tauri and red blood cells occur in the absence of transcription (O'Neill and Reddy, 2011, O'Neill et al., 2011), reveal that other circadian timekeeping mechanisms exist in eukaryotes that do not require transcriptional feedback. I discuss the role of circadian feedback loops in circadian timekeeping in light of these new results.
Section snippets
The Circadian Feedback Loops of Drosophila
The per feedback loop suggested a mechanism for keeping circadian time: once per transcription is initiated during mid-day, the levels of per mRNA rise until early evening, when accumulating levels of PER protein repress per transcription, thereby reducing the levels of per mRNA until the early day, when PER protein is eliminated and the next round of per transcription begins. Although this feedback loop provided a framework for how per contributes to circadian timekeeping, it raised many
Posttranscriptional Regulation of Rhythmic Transcription
To keep circadian time, the various molecular events within the core feedback loop must be completed in ∼ 24 h. However, the transcriptional activation and elongation, transcript processing and transport to the cytosol, protein synthesis and accumulation, nuclear localization, transcriptional repression, and repressor degradation that mediate feedback loop function collectively take much less than 24 h to complete. Consequently, delays must be imposed at one or more steps in the core loop to
Light Entrainment of the Drosophila Circadian Feedback Loops
In animals, environmental cycles of light, temperature, and/or social cues set the phase of (i.e., entrain) circadian oscillators so that overt rhythms in physiology, metabolism, and behavior occur at the appropriate time of day. The most potent and reliable environmental cue is light, which mediates entrainment via different mechanisms depending on tissue type and species. In mammals, light is detected by melanopsin in retinal ganglion cells in the eye, which transmit signals to the “master
Summary and Conclusions
In this chapter, I have covered several topics related to the interlocked feedback loops that keep circadian time in Drosophila. There are several take home points from this review. First, many feedback loop components have been identified in Drosophila that carry out specific roles in regulating time-dependent transcription including the CLK and CYC activators and PER and TIM repressors. Although the per and Clk feedback loops drive rhythmic transcription in opposite phases of the circadian
Acknowledgments
I want to thank Dr. Wangjie Yu for comments on the chapter and Isaac Edery for communicating unpublished results. This work was supported by NIH Grant NS052854.
References (179)
- et al.
A mutant Drosophila homolog of mammalian Clock disrupts circadian rhythms and transcription of period and timeless
Cell
(1998) - et al.
New short period mutations of the Drosophila clock gene per
Neuron
(1992) - et al.
Cycling vrille expression is required for a functional Drosophila clock
Cell
(1999) - et al.
NEMO/NLK primes phosphorylation of a time-delay phospho-cluster on PERIOD revealing a novel mechanism for how circadian clock speed is set
Cell
(2011) - et al.
Circadian biology: Environmental regulation of a multi-oscillator network
Curr. Biol.
(2010) - et al.
Circadian regulation of gene expression systems in the Drosophila head
Neuron
(2001) - et al.
Drosophila CRYPTOCHROME is a circadian transcriptional repressor
Curr. Biol.
(2006) - et al.
Temporally regulated nuclear entry of the Drosophila period protein contributes to the circadian clock
Neuron
(1995) - et al.
vrille, Pdp1, and dClock form a second feedback loop in the Drosophila circadian clock
Cell
(2003) - et al.
How a cyanobacterium tells time
Curr. Opin. Microbiol.
(2008)
Molecular bases for circadian clocks
Cell
CRY, a Drosophila clock and light-regulated cryptochrome, is a major contributor to circadian rhythm resetting and photosensitivity
Cell
Drosophila CRY is a deep brain circadian photoreceptor
Neuron
A promoterless period gene mediates behavioral rhythmicity and cyclical per expression in a restricted subset of the Drosophila nervous system
Neuron
Nocturnal male sex drive in Drosophila
Curr. Biol.
VRILLE feeds back to control circadian transcription of Clock in the Drosophila circadian oscillator
Neuron
The circadian timekeeping system of Drosophila
Curr. Biol.
Essential and expendable features of the circadian timekeeping mechanism
Curr. Opin. Neurobiol.
The circadian clock of fruit flies is blind after elimination of all known photoreceptors
Neuron
Regulation of the Drosophila protein timeless suggests a mechanism for resetting the circadian clock by light
Cell
Drosophila CLOCK protein is under posttranscriptional control and influences light-induced activity
Neuron
Positional cloning of the mouse circadian clock gene
Cell
The Drosophila clock gene double-time encodes a protein closely related to human casein kinase Iepsilon
Cell
Phosphorylation of period is influenced by cycling physical associations of double-time, period, and timeless in the Drosophila clock
Neuron
Spike amplitude of single-unit responses in antennal sensillae is controlled by the Drosophila circadian clock
Curr. Biol.
Social experience modifies pheromone expression and mating behavior in male Drosophila melanogaster
Curr. Biol.
mCRY1 and mCRY2 are essential components of the negative limb of the circadian clock feedback loop
Cell
The Drosophila CLOCK protein undergoes daily rhythms in abundance, phosphorylation, and interactions with the PER-TIM complex
Neuron
Posttranslational mechanisms regulate the mammalian circadian clock
Cell
HY4 gene of A. thaliana encodes a protein with characteristics of a blue-light photoreceptor
Nature
A role for CK2 in the Drosophila circadian oscillator
Nat. Neurosci.
Circadian organization of behavior and physiology in Drosophila
Annu. Rev. Physiol.
Circadian regulation of a Drosophila homolog of the mammalian Clock gene: PER and TIM function as positive regulators
Mol. Cell. Biol.
dCLOCK is present in limiting amounts and likely mediates daily interactions between the dCLOCK-CYC transcription factor and the PER-TIM complex
J. Neurosci.
Molecular genetics of a biological clock in Drosophila
Proc. Natl. Acad. Sci. USA
Restoration of circadian behavioural rhythms by gene transfer in Drosophila
Nature
Changes in abundance or structure of the per gene product can alter periodicity of the Drosophila clock
Nature
Circadian rhythms from multiple oscillators: Lessons from diverse organisms
Nat. Rev. Genet.
PDP1epsilon functions downstream of the circadian oscillator to mediate behavioral rhythms
J. Neurosci.
The blue-light photoreceptor CRYPTOCHROME is expressed in a subset of circadian oscillator neurons in the Drosophila CNS
J. Biol. Rhythms
The circadian output gene takeout is regulated by Pdp1epsilon
Proc. Natl. Acad. Sci. USA
Preliminary action spectra suggest that the clock cells of Drosophila are synchronized to the external LD-cycle by the compound eyes plus extraretinal photoreceptors
Integration of light and temperature in the regulation of circadian gene expression in Drosophila
PLoS Genet.
Roles of the two Drosophila CRYPTOCHROME structural domains in circadian photoreception
Science
Light-dependent sequestration of TIMELESS by CRYPTOCHROME
Science
Genome-wide expression analysis in Drosophila reveals genes controlling circadian behavior
J. Neurosci.
Clock-gated photic stimulation of timeless expression at cold temperatures and seasonal adaptation in Drosophila
J. Biol. Rhythms
Thermosensitive splicing of a clock gene and seasonal adaptation
Cold Spring Harb. Symp. Quant. Biol.
Interlocked feedback loops contribute to the robustness of the Neurospora circadian clock
Proc. Natl. Acad. Sci. USA
The phospho-occupancy of an atypical SLIMB-binding site on PERIOD that is phosphorylated by DOUBLETIME controls the pace of the clock
Genes Dev.
Cited by (286)
Identification and characterization of circadian clock genes in the head transcriptome of Conopomorpha sinensis Bradley
2024, Comparative Biochemistry and Physiology - Part D: Genomics and ProteomicsClocks at sea: the genome-editing tide is rising
2024, Trends in GeneticsAngelica sinensis polysaccharide extends lifespan and ameliorates aging-related diseases via insulin and TOR signaling pathways, and antioxidant ability in Drosophila
2023, International Journal of Biological MacromoleculesCircadian clock genes and photoperiodic diapause in the moth Sesamia nonagrioides
2023, Comparative Biochemistry and Physiology Part - B: Biochemistry and Molecular BiologyBehavioral circatidal rhythms require Bmal1 in Parhyale hawaiensis
2023, Current Biology