BIOLOGICAL OVERVIEW

In Drosophila, there are two well-characterized photoperiod response genes: period (per) and timeless (tim). The protein levels, RNA levels, and transcription rates of these two genes undergo robust circadian oscillations. In addition, mutations in the two proteins (Per and Tim) alter or abolish the periodicity and phase of these rhythms, demonstrating that both proteins regulate their own transcription. Although there is no evidence indicating that the effects on transcription are direct, Per contains a PAS domain, which has been shown to mediate interactions between transcription factors. Most of these PAS-containing transcription factors also contain well-characterized basic helix-loop-helix (bHLH) DNA-binding domains. However, Per lacks any known DNA-binding domain, and there is no evidence that Per interacts directly with DNA. Therefore it was proposed that Per regulates transcription by interacting with DNA-binding transcription factors of the bHLH-PAS family and how Per transcription is regulated remained an open question (Allada, 1998 and references).

Recent data have extended this model in two ways: (1) an enhancer has been identified in the per promoter capable of driving cycling transcription of a reporter gene (Hao, 1997). Notably, the activity of this 69-base pair element requires an E box (CACGTG), a known binding site for some bHLH transcription factors, including bHLH-PAS transcription factors. (2) The cloning of the mouse circadian rhythm gene, mClock, revealed a bHLH-PAS transcription factor involved in circadian rhythms. Recently, mouse per genes have been identified and found to undergo circadian oscillation in mammalian clock tissues. Thus, mouse CLOCK may drive the cycling transcription of mouse per genes through evolutionarily conserved E box elements in mouse per promoters. If so, one might expect to find a Drosophila orthologs of mClock, which would drive cycling of the Drosophila per gene (Allada, 1998).

Jrk, a novel arrhythmic Drosophila mutant, has been identified which severely disrupts cycling transcription of the per and tim genes. The cloning and identification of the Jrk gene reveals that it is the apparent homolog of the mouse Clock gene; it has therefore been named Drosophila Clock (Clk). The mutation in Clk results in a premature stop codon that truncates the protein, deleting most of the putative C-terminal activation domain. This truncation is consistent with the semidominant mutant phenotype, similar to the original mouse Clock mutant (Allada, 1998).

A second Drosophila clock gene, cycle (cyc) has also been cloned. Homozygous mutant cyc flies have a behavioral and molecular phenotype that resembles closely that of homozygous Clk flies: they are behaviorally arrhythmic and exhibit little per and tim transcription. Further phenogenetic analyses indicate that, like Clk, the cyc locus has a dosage effect on period. It is suggested that cycle is a nonvital, dedicated clock gene. Cloning of cyc indicates that, like Clk, it encodes a bHLH-PAS transcription factor and is a Drosophila homolog of the human gene BMAL1 (MOP3) (Ikeda, 1997; Hogenesch, 1997 and 1998). Biochemical work (Hogenesch, 1998) indicates that BMAL1 is the partner of mammalian CLOCK and that the heterodimer binds to and activates transcription from per-like E boxes. Based on all of these results, it is proposed that the CYC:CLK heterodimer binds to per and tim E boxes and makes a major contribution to the circadian transcription of Drosophila clock genes (Rutila, 1998). Further characterization of the Drosophila Clk mutant phenotype and a second study (Hogenesch, 1998) suggest that the wild-type Drosophila protein (Clk) interacts directly with the per and tim E boxes and makes a major contribution to the circadian transcription of clock genes. The similar mouse mutant phenotype and the remarkable sequence conservation strongly support the presence of similar clock mechanisms and components in the common ancestor of Drosophila and mammals more than 500 million years ago (Allada, 1998).

Placed in the context of the current understanding of the Drosophila circadian oscillator, these results indicate that Clock closes the feedback loop. A Clock-Cycle complex drives expression of per and tim by binding an E-box that is present in their promoters. With time, Per and Tim heterodimers accumulate, translocate to the nucleus, and act as dominant negative inhibitors of Clock-Cycle. As mRNA and protein levels fall, the inhibition is relieved, which allows Clock-Cycle to initiate a new round of synthesis (Darlington, 1998).

Drosophila Clock (Clk) is rhythmically expressed, with peaks in mRNA and protein (Clk) abundance early in the morning. Clk mRNA cycling is regulated by Period-Timeless (Per-Tim)-mediated release of Clk- and Cycle (Cyc)-dependent repression. Lack of both Per-Tim derepression and Clk-Cyc repression results in high levels of Clk mRNA, which implies that a separate Clk activator is present. These results demonstrate that the Drosophila circadian feedback loop is composed of two interlocked negative feedback loops: a per-tim loop, which is activated by Clk-Cyc and repressed by Per-Tim, and a Clk loop, which is repressed by Clk-Cyc and derepressed by Per-Tim (Glossop, 1999).

Comparatively little is known about the regulation of Clk mRNA cycling. The levels of Clk mRNA are low in mutants lacking Per (per⁰¹) or Tim (tim⁰¹) function, which suggests that Per and Tim activate Clk transcription in addition to their roles as transcriptional repressors. The mechanism of Per-Tim-dependent activation is not known, but three models have been proposed to account for this activation. In the first two models, Per and Tim promote Clk transcription by shuttling transcriptional activators into the nucleus or by coactivating a transcriptional complex. In the third model, Per or Tim or both inhibit the activity of a transcriptional repressor complex (Glossop, 1999).

To distinguish among these alternative models, Clk mRNA levels were measured in different clock gene mutant combinations. Because Clk and Cyc are both required for per and tim activation, it was predicted that mutants lacking functional Clk (Clk^Jrk) or Cyc (Cyc⁰) would exhibit low levels of Clk mRNA because the concentrations of the Per and Tim activators (of Clk) would be low. It was surprising to find that the level of Clk mRNA is indistinguishable from the wild-type peak in both mutants. The levels of Clk mRNA do not vary significantly over the circadian cycle in these mutants, which is consistent with the lack of a functional circadian oscillator (Glossop, 1999).

The high level of Clk mRNA in the absence of Clk-dependent Per accumulation indicates that Per-dependent Clk activation does not occur by nuclear localization of an activator or by coactivation. However, the possibility remains that low levels of per and tim transcripts in Clk^Jrk or Cyc⁰ mutants lead to some active Per-Tim dimer formation and subsequent activation of Clk transcription. To eliminate this possibility, Clk mRNA levels were measured in per⁰¹;Clk^Jrk and per⁰¹;Cyc⁰ double mutants. In both cases, the levels of Clk mRNA observed under light-dark (LD) or constant dark (DD) conditions are close to the peak level in wild-type flies, indicating that Per-Tim activates Clk transcription through derepression (Glossop, 1999).

The Clk repressor that is removed as a result of Per-Tim accumulation appears to be either Clk-Cyc itself or a repressor that is activated by Clk-Cyc. When comparing the levels of Clk between per⁰¹ flies and per⁰¹;Clk^Jrk or per⁰¹;Cyc⁰ double mutants, the presence of active Clk and Cyc results in the repression of Clk transcript accumulation. In per⁰¹ mutants, Clk mRNA is at low but detectable levels. This suggests that in the absence of Per-Tim derepression, Clk transcription reaches a steady state in which activation and Clk-Cyc-dependent repression equilibrate to produce low levels of Clk mRNA transcripts and, hence, of Clk protein. In per⁰¹ and tim⁰¹ mutants, per and tim transcription is constitutive and per and tim transcripts are relatively low in abundance. This result can be explained by the partial activation of per and tim by low levels of Clk-Cyc dimers in the absence of Per-Tim repression (Glossop, 1999).

On the basis of these observations, it is proposed that interlocked negative feedback loops mediate circadian oscillator function in Drosophila (see reviews by Hardin, The Circadian Timekeeping System of Drosophila and Vallone, Start the clock! Circadian rhythms and development). Late at night, Per-Tim dimers in the nucleus bind to and sequester Clk-Cyc dimers. This interaction effectively inhibits Clk-Cyc function, which leads to the repression of per and tim transcription and the derepression of Clk transcription. As Per-Tim levels fall early in the morning (ZT 0-3), Clk-Cyc dimers are released and repress Clk expression, thereby decreasing Clk mRNA levels so that they are low by the end of the day (ZT 12). Concomitant with the drop in Clk mRNA levels (through Clk-Cyc-dependent repression) is the accumulation of per and tim mRNA (through E-box-dependent Clk-Cyc activation). As Clk mRNA falls to low levels early in the evening (ZT 15), the levels of Clk-Cyc also fall, leading to a decrease in per and tim transcription and an increase in Clk mRNA accumulation. A new cycle then begins as high levels of Per and Tim enter the nucleus and Clk starts to accumulate late at night (Glossop, 1999).

These observations also fit well with the regulation of Drosophila cryptochrome (cry), whose mRNA cycles in phase with that of Clk. Like Clk, CRY mRNA transcripts are constitutively low in per⁰¹ mutants and constitutively high in Clk^Jrk or Cyc⁰ single mutants and in per⁰¹;Clk^Jrk or per⁰¹;Cyc⁰ double mutants. These striking similarities between Clk and CRY mRNA phases (in the wild type) and Clk and CRYmRNA levels in circadian mutants suggest that the cry locus may be regulated by the same Per-Tim release of Clk-Cyc repression mechanism as Clk (Glossop, 1999).

These results reveal the existence of a Clk feedback loop and its regulatory interactions with the well-characterized per-tim feedback loop. One clear prediction from these experiments is that there is a separate activator of Clk expression. Such an activator is indicated by the high levels of Clk mRNA in the absence of Per and of either Clk or Cyc. This observation is somewhat surprising because the presence of this activator is independent of factors that control the expression of other clock genes (that is, Per, Clk, and Cyc) (Glossop, 1999).

Data supporting the existence of interlocked per-tim and Clk feedback loops were obtained from whole heads, raising the possibility that Clk expression in small subsets of 'clock-specific' cells such as the locomotor activity pacemaker cells (that is, lateral neurons) could be masked by Clk expression in other tissues. However, the autonomy and synchrony of per expression in diverse tissues in the head and body suggest that the circadian feedback loop mechanism is the same in all tissues and argue against fundamental tissue-specific differences in the feedback loop mechanism (Glossop, 1999).

An important aspect of circadian biology is how the clock regulates clock-controlled genes (CCGs). In mammals, it has been shown in vitro that CLOCK and BMAL-1 (the mammalian ortholog of Cyc) activate vasopressin gene transcription and that all three mouse Pers and Tim repress this activation, resulting in peak vasopressin mRNA transcripts by midmorning (ZT 6). Although this mode of regulation may be more general for CCGs whose mRNA transcripts peak in phase with per (or mPer), it does not explain how CCGs that cycle in antiphase are regulated. The results presented here provide a possible mechanism by which the clock regulates CCGs whose mRNAs cycle in antiphase to those of per. The similarities between Clk and cry mRNA profiles in the wild type and in several single and double circadian mutants suggest that Per-Tim release of Clk-Cyc repression may serve a more general role in regulating CCG mRNAs that cycle in antiphase to per mRNA (Glossop, 1999).

GENE STRUCTURE

Within a Drosophila Clock intron, a second ORF is found with significant similarity to the mammalian gene EB1, implicated in binding the adenomatosis polyposis coli (APC) C terminus (Allada, 1998).

PROTEIN STRUCTURE

Amino Acids - 1015 (Allada, 1998); 1023 (Darlington, 1998)

Structural Domains

Sequencing of multiple cDNAs indicates one open reading frame. The ORF has at least two forms: one corresponds to the full-length protein of 1015 amino acids and the other to a protein missing the bHLH and PAS A regions. Several transcript forms are found, none of which demonstrate robust circadian oscillations. The full-length Drosophila Clock protein contains all of the known subregions of mouse Clock, including bHLH, PAS A, PAS B, and prominent Q-rich activation domain (Allada, 1998).

Alignment of the full-length Drosophila Clock protein with mouse Clock reveals 35% identity over the entire overlap (>800 amino acids). Drosophila Clock also shows substantial sequence similarity to the mouse and human bHLH-PAS proteins NPAS2/MOP4, but the latter has no polyQ regions. There is no functional evidence, however, linking NPAS2/MOP4 to circadian rhythms. The sequence identity of Drosophila and Mouse Clock proteins is even more impressive in the three subregions where one can infer a biochemical function. The bHLH domains, involved in DNA binding and protein dimerization, have 71% similarity and 60% identity. The basic region, involved in sequence-specific DNA contacts, is remarkably conserved with 11 out of 13 amino acids being identical; this suggests that the two proteins bind to similar if not identical DNA targets. In fact, 6 out of 9 amino acids are identical to a consensus generated for bHLH proteins that bind the CAC/GTG E box half-site, including the critical R residue at position 15 (Allada, 1998).

The PAS region, implicated in protein dimerization, is also strikingly conserved between the insect and murine genes. The PAS B repeat and the region within PAS just C-terminal to PAS B are particularly conserved; 79% identical and 91% similar over a span of 107 amino acids (amino acids 262-368). The conservation of PAS and its demonstrated role in dimerization suggest that Drosophila Clock and mouse Clock may have conserved heterodimeric partners. Indeed, it appears that another Drosophila clock gene, cycle, encodes the relevant bHLH-PAS partner and that the same heterodimeric complex is functionally relevant in both systems. The third conserved region is the glutamine (Q)-rich C terminus of the protein. Glutamine-rich regions, especially polyglutamine repeats, are known to function in transcriptional activation (Allada, 1998).

date revised: 25 February 2000

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.