Molecular rhythms are generated by transcriptional- transcriptional-translational feedback regulation

Molecular circadian rhythm research began with the discovery of the clock gene period in 1971 (Konopka and Benzer 1971). After treatment with the mutagen ethyl methane sulfonate, Konopka and Benzer could isolate three mutant fly lines showing severe impairments of circadian behavior (Fig. 4). One mutant was arhythmic (per⁰), one showed a shortened period length of 19 h (per^s), and the third showed a longer period length of 28 h (per^l). All three mutations could be traced to one gene located on the X chromosome, which was termed period (per). Remarkably, these were the first experiments showing that mutations of a single gene can affect behavior (Konopka and Benzer 1971).

Later the per⁰ and per^s mutation could be mapped to single nucleotides of the per gene. A single nucleotide substitution resulted in a nonsense mutation in the case of per⁰ and a missense mutation in the case of per^s (Yu et al. 1987). More than 20 years later the next clock gene, timeless (tim), was discovered (Sehgal et al. 1994). Then, in closer succession dClock (clk, Allada et al. 1998), cycle (cyc,

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Rutila et al. 1998), cryptochrome (dcry, in this thesis termed cry1, Emery et al. 1998; Stanewsky et al.

1998), double-time (dbt, Kloss et al. 1998; Price et al. 1998), vrille (vri, Blau and Young 1999), timeless 2 (timeout, tim2, Benna et al. 2000), par domain protein 1ε (pdp1ε, McDonald and Rosbash 2001), shaggy (sgg, Martinek et al. 2001), casein kinase 2 (ck2, Lin et al. 2002b), supernumerary limbs (slimb, Ko et al. 2002), protein phosphatase 2A (pp2A, Sathyanarayanan et al. 2004), jetlag (jet, Koh et al. 2006; Peschel et al. 2006), protein phosphatase 1 (pp1, Fang et al. 2007), clockwork-orange (cwo, Kadener et al. 2007; Lim et al. 2007; Matsumoto et al. 2007), and twenty-four (tyf, Lim et al.

2011) were found (Tab. 1).

Fig. 4. Mutations in the period gene strongly affect the period of locomotor activity. A-D. The locomotor activity (symbolized by black bars) of four individual fruit flies with different genotypes is shown for five consecutive days in constant darkness. The fly with the wildtype period gene (per^WT) has a period length of 23.5 h (A), while the fly carrying the per^s mutation has a shortened period length of 19.5 h (B), and the fly with the per^l mutation a long period length of 28 h (C). The fly with the per⁰ mutation does not show rhythmic locomotor activity (D). Redrawn and modified after Konopka and Benzer (1971).

Molecular rhythms in the clock cells are generated via different interconnected transcriptional-translational feedback loops, in which positive elements activate the expression of negative elements, which feed back and repress the action of the positive elements (Sandrelli et al. 2008, Fig. 5). In the core feedback loop the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) transcription factors CLK and CYC form a heterodimer (CLK:CYC) and activate transcription of the genes per and tim via binding to the genes' E-boxes (Hao et al. 1997; Allada et al. 1998; Darlington et al. 1998; Rutila et al. 1998). The proteins PER and TIM accumulate in the cytoplasm and form a complex with the kinase DBT, which translocates into the nucleus (Curtin et al. 1995; Gekakis et al. 1995; Kloss et al.

1998; Kloss et al. 2001). Here, PER mediates DBT-dependent phosphorylation of CLK leading to a repression of E-box binding and thus to an inhibition of PER's and TIM's "own transcription" (Lee et al. 1999; Kim and Edery 2006; Yu et al. 2006). Therefore, an oscillation in the mRNA and protein levels of per and tim is generated (Hardin et al. 1990; Zerr et al. 1990; Sehgal et al. 1995; Zeng et al.

1996).

Like CLK and CYC, PER also contains a PAS domain, which appears to be required for dimerization of PER and TIM or CLK and CYC (Huang et al. 1993). The functional significance of the PER-TIM-dimerization with respect to the nuclear entry is not yet clear. In different studies the heterodimers were shown to dissociate before nuclear entry (Meyer et al. 2006), PER and TIM were shown to accumulate in the nucleus with different kinetics (Shafer et al. 2002; Rieger et al. 2006), and PER was shown to be a more potent inhibitor of CLK:CYC-mediated transcription, if TIM was absent (Rothenfluh et al. 2000). However, in other studies TIM was shown to be required for nuclear entry of PER (Saez and Young 1996; Saez et al. 2011), and TIM protected PER from degradation and thus plays an important role for stabilization of PER (Kloss et al. 2001). For both, PER and TIM a circadian rhythm in their phosphorylation status was shown (Edery et al. 1994; Zeng et al. 1996), which

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appears to be the major determinant for nuclear entry and stability, with hypophosphorylated forms being more stable than hyperphosphorylated forms, which precede and most probably cause degradation (Price et al. 1998; Kloss et al. 2001; Martinek et al. 2001; Stoleru et al. 2007). Thus, several kinases and phosphatases balance the phosphorylation status of the feedback loop components (Tab. 1). Apparently these posttranslational modifications allow for a characteristic delay of up to eight hours between peak levels of per and tim mRNA and protein, which is a prerequisite for the period length of 24 h (Hunter-Ensor et al. 1996; Hamilton and Kay 2008; Hardin 2011). Additionally, translational control might contribute to this delay. For example, the lateral but not the dorsal clock neurons express TYF (Lim et al. 2011), which was shown to interact with per and tim mRNA and translational components such as the 5'-cap-binding complex and poly(A)-binding protein, suggesting that TYF is a translational activator. Probably translation of PER and TIM is first inhibited by yet unidentified translation repressors and then promoted by translational activators such as TYF (Lim et al. 2011).

Next to the core feedback loop other interlocked feedback loops exist (Fig. 5). Not only expression of PER and TIM, but also expression of the basic leucine zipper (bZIP) transcription factors VRI and PDP1ε are regulated by the CLK:CYC heterodimer (Blau and Young 1999; McDonald and Rosbash 2001). Vri mRNA and protein levels peak at ZT 14, at about the same time as per and tim mRNA levels, while peak levels of pdp1ε are delayed (mRNA: ZT 18, protein: ZT 21, Review: Hamilton and Kay 2008). Both proteins translocate into the nucleus and bind to specific sites in the clk promoter.

While clk transcription is inhibited by VRI binding, it is activated several hours later by PDP1ε binding (Cyran et al. 2003; Glossop et al. 2003), resulting in oscillations of clk mRNA with peak levels around ZT 0 (Review: Hamilton and Kay 2008). In contrast to CLK, the gene of its dimerization partner CYCLE is not rhythmically expressed, although its name may suggest otherwise (Rutila et al. 1998). While one study could show circadian oscillations in the CLK protein levels employing Western blots (Lee et al. 1998), another study employing immunohistochemistry found constitutive high CLK levels throughout the day (Houl et al. 2006), indicating a non-identified posttranscriptional or posttranslational regulation mechanism leading from rhythmic mRNA levels to constant protein levels. However, regulation of CLK:CYC mediated transcription certainly depends on CLK's phosphorylation status with hyperphosphorylated CLK shown to be degraded (Kim and Edery 2006;

Yu et al. 2006).

In addition to VRI and PDP1 the bHLH orange-domain transcription factor CWO was shown to be CLK:CYC-dependently expressed and to exert negative feedback (Fig. 5). Cwo mRNA reaches its peak levels around ZT 12 - 15 (Hamilton and Kay 2008) and the protein represses CLK:CYC-mediated transcription via binding to its target genes' E-boxes including cwo itself. Thus, CWO competes with CLK for the binding site, apparently leading to high amplitude oscillations of the target genes (Kadener et al. 2007; Lim et al. 2007; Matsumoto et al. 2007). However, another study found an inhibitory effect of CWO on its own transcription but activating effects on transcription of per, tim, vri, and pdp1 (Richier et al. 2008). It was suggested that CWO could activate transcription in the evening when the nuclear PER level is low, and inhibit transcription in the morning, when the nuclear PER level is high (Richier et al. 2008).

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Tab. 1. Clock genes of the fruit fly D. melanogaster (modified after Hamilton and Kay 2008)

Clock gene Expression Characteristics Reference

Core loop

cycle (cyc) constitutive See clk (Rutila et al. 1998)

dClock (clk) rhythmic bHLH-PAS-transcription factor, dimerization with CLK, activates transcription of per, tim, pdp1, and vrille via E-box binding

(Allada et al. 1998; Bae et al. 1998;

Darlington et al. 1998; Lee et al. 1998, 1999; Houl et al. 2006)

period (per) rhythmic Dimerization with TIM via PAS-domain, inhibits CLK-CYC-mediated transcription

(Konopka and Benzer 1971; Yu et al.

1987; Hardin et al. 1990; Edery et al.

1994; Gekakis et al. 1995; Hao et al.

1997; Meyer et al. 2006)

timeless (tim) rhythmic Dimerization with PER, stabilization of PER, target of CRY1-dependent light input

(Sehgal et al. 1994; Sehgal et al. 1995;

Hunter-Ensor et al. 1996; Myers et al.

1996; Zeng et al. 1996; Naidoo et al.

1999)

Interconnected loops

clockwork orange (cwo)

rhythmic bHLH orange-domain transcription factor, inhibition (activation) of CLK-CYC mediated transcription via E-box binding

(Kadener et al. 2007; Lim et al. 2007;

Matsumoto et al. 2007; Richier et al.

2008)

par domain protein 1ε (pdp1ε)

rhythmic bZIP transcriptions factor, activation of clk (and possibly cry1) transcription, clk-independent regulation of circadian output

(McDonald and Rosbash 2001; Cyran et al. 2003; Benito et al. 2007; Zheng et al. 2009)

vrille (vri) rhythmic bZIP transcriptions factor, repression of clk and cry1 transcription

(Blau and Young 1999; Cyran et al.

2003; Glossop et al. 2003)

Regulators

casein kinase 2 (ck2)

? Phosphorylation of PER and TIM, promotion of nuclear translocation of PER and TIM

(Lin et al. 2002b; Akten et al. 2003; Lin et al. 2005; Meissner et al. 2008)

cryptochrome (dcry/cry1)

rhythmic Blue-light photoreceptor expressed in pacemaker cells, light-entrainment, interaction with PER, TIM, and JET,

mediation of light-dependent depolarization in a TIM-independent manner, function in the core feedback loop in peripheral clocks.

(Emery et al. 1998; Stanewsky et al.

1998; Ceriani et al. 1999; Emery et al.

2000a; Emery et al. 2000b; Krishnan et al. 2001; Lin et al. 2001; Rosato et al. 2001; Cyran et al. 2003; Collins et al. 2006; Yoshii et al. 2008; Fogle et al.

2011)

double-time (dbt)

constitutive Phosphorylation of PER and CLK, promotion of PER- and CLK-degradation

(Kloss et al. 1998; Price et al. 1998;

Kim and Edery 2006; Yu et al. 2006)

jetlag (jet) ? F-box protein, transmission from light-signals from CRY1 to TIM, targeting TIM for degradation

(Koh et al. 2006; Peschel et al. 2006)

protein phosphatase 1 (pp1)

? Dephosphorylation of TIM, stabilization of TIM (and PER)

(Fang et al. 2007)

protein phosphatase 2A (pp2A)

? Dephosphorylation of PER and CLK, promotion of PER-stability and nuclear translocation

(Sathyanarayanan et al. 2004; Kim and Edery 2006; Fang et al. 2007)

shaggy (sgg) constitutive Glycogen synthase kinase-3 (GSK-3) orthologue, regulation PER-TIM nuclear translocation via phosphorylation of TIM, interaction with CRY1

(Martinek et al. 2001; Stoleru et al.

2007)

supernumerary limbs (slimb)

? F-box/WD40-repeat protein, component of the ubiquitin-proteasome pathway, targeting PER for degradation

(Ko et al. 2002)

timeless 2 (tim2)

? Mammalian TIM orthologue, requirement for chromosome integrity, residual function in light-entrainment

(Benna et al. 2000; Benna et al. 2010)

twenty-four (tyf)

? Promotion of PER translation (Lim et al. 2011)

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Fig. 5. Transcriptional-translational feedback regulation in central clock neurons of D. melanogaster. Central elements of the interconnected feedback loops are the transcription factors CLOCK (CLK) and CYCLE (CYC), which activate transcription of the genes period (per), timeless (tim), vrille (vri), par domain protein 1 ε (pdp1), and clockwork orange (cwo). PER and TIM form a complex with the kinase DOUBLETIME (DBT), which translocates to the nucleus and inhibits CLK/CYC-mediated transcription. TWENTY-FOUR (TYF) promotes translation of per and tim mRNA. Next to DBT, the kinases SHAGGY (SGG) and CASEIN KINASE 2 (CK2) and the PROTEIN PHOSPHATASES 1 and 2A (PP1 and PP2A) regulate the phosphorylation status of the PER/TIM complex. In the second loop VRI inhibits, while PDP1 activates transcription of clk by binding to the gene's V/P-box. In the third loop CWO inhibits CLK/CYC-mediated transcription. Redrawn and modified after (Tomioka et al.

2012).

Im Dokument Physiological analysis of central and peripheral insect circadian pacemaker neurons (Seite 32-36)