Elsevier

Advances in Genetics

Volume 74, 2011, Pages 141-173
Advances in Genetics

Chapter 5 - Molecular Genetic Analysis of Circadian Timekeeping in Drosophila

https://doi.org/10.1016/B978-0-12-387690-4.00005-2Get rights and content

Abstract

A genetic screen for mutants that alter circadian rhythms in Drosophila identified the first clock gene—the period (per) gene. The per gene is a central player within a transcriptional feedback loop that represents the core mechanism for keeping circadian time in Drosophila and other animals. The per feedback loop, or core loop, is interlocked with the Clock (Clk) feedback loop, but whether the Clk feedback loop contributes to circadian timekeeping is not known. A series of distinct molecular events are thought to control transcriptional feedback in the core loop. The time it takes to complete these events should take much less than 24 h, thus delays must be imposed at different steps within the core loop. As new clock genes are identified, the molecular mechanisms responsible for these delays have been revealed in ever-increasing detail and provide an in-depth accounting of how transcriptional feedback loops keep circadian time. The phase of these feedback loops shifts to maintain synchrony with environmental cycles, the most reliable of which is light. Although a great deal is known about cell-autonomous mechanisms of light-induced phase shifting by CRYPTOCHROME (CRY), much less is known about non-cell autonomous mechanisms. CRY mediates phase shifts through an uncharacterized mechanism in certain brain oscillator neurons and carries out a dual role as a photoreceptor and transcription factor in other tissues. Here, I review how transcriptional feedback loops function to keep time in Drosophila, how they impose delays to maintain a 24-h cycle, and how they maintain synchrony with environmental light:dark cycles. The transcriptional feedback loops that keep time in Drosophila are well conserved in other animals, thus what we learn about these loops in Drosophila should continue to provide insight into the operation of analogous transcriptional feedback loops in other animals.

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.

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