CLK and CYC are transcription factors containing a PAS (PER--ARNT--SIM) dimerization domain and a basic helix--loop--helix (bHLH) domain involved in DNA binding. If one of them is mutated, flies lose their eclosion and locomotor rhythms (Allada et al., 1998; Rutila et al., 1998). Therefore, they are crucial clock components. Moreover, Clk and cyc mutants negatively affect the expression levels of other clock genes like per, tim and vri (Allada et al., 1998; Rutila et al., 1998; Blau and Young, 1999), thus CLK and CYC positively influence transcription of other genes. Indeed, the CLK:CYC heterodimer binds to the E--box sequences--a stretch of six consensus nucleotides recognized by bHLH transcription factors (Kyriacou and Rosato, 2000)--in the promoters of per and tim and activates their transcription (Darlington et al., 1998; Lee et al., 1999).
4.2.2 Regulation of transcriptional activators by PER, TIM, DBT and SGG The PER protein is a founding member of the PAS proteins, but lacks the bHLH DNA--binding domain. PER forms a dimer with TIM (Gekakis et al., 1995). TIM is not a PAS protein but it contains three ARMADILLO--like dimerization domains and two of them are meant to act as PER binding sites (Kyriacou and Hastings, 2001; Saez and Young, 1996). The PER:TIM heterodimer moves into the nucleus and binds to the CLK:CYC heterodimer, which stops per and tim transcription by interfering with the functioning of CLK:CYC on the per and tim promoters (Fig. 3;
Lee et al., 1998, 1999; Bae et al., 2000). However, there is evidence that PER can function on its own, as it alone could move into the nucleus (Shafer et al., 2002)..
The PER protein is phosphorylated by the DOUBLE--TIME (DBT) kinase in the cytoplasm (Kloss et al., 1998, Price et al., 1998), which renders PER unstable until it dimerizes with TIM. The SHAGGY (SGG) phosphorylates TIM (Fig. 3), and thus SGG regulates the nuclear entry of TIM (Martinek et al., 2001). The nuclear entry of PER is similarly regulated by DBT (Bao et al., 2001).
CLK CYC
ClkRNA peaks – lights on CLKprotein vriRNA peaks end of day
PDP 1protein
ClkRNA peaks – lights on CLKprotein vriRNA peaks end of day
PDP 1protein
Figure 3 Schematic view of the circadian clock work mechanism in Drosophila. It is based on the interconnected feed--back loops. In the first loop, the CLOCK:CYCLE (CLK:CYC) dimer complex binds to the E--boxes (represented by multiple E) in the per and tim promoter, thus positively control their transcription. In early/midnight, per and tim RNA levels peak in the cytoplasm. During the late night PERIOD (PER) and TIMELESS (TIM)
accumulate in the cytoplasm, subsequently they are phosphorylated by DOUBLE--TIME (DBT) and SHAGGY (SGG), respectively. The phosphorylated PER and TIM (with a P attached in the picture) form the heterodimer PER:TIM. In the late night, PER:TIM shuttles into the nucleus and binds to CLK:CYC, which closes the loop. The binding of PER:TIM to CLK:CYC stops the transcriptional activation of per and tim, thus achieves a negative feed--back on their own production levels.
In the second loop, again CLK:CYC positively activates the transcription of vrille (vri) and Par domain protein 1 (Pdp 1). Due to their slight temporal difference in transcription vri RNA peaks in the end of day but Pdp 1 RNA peaks in the midnight. Correspondingly, VRILLE (VRI) peaks in the early night and PAR DOMAIN PROTEIN 1 (PDP 1) peaks in the midnight. When CLK levels are high during the late day, by then VRI level rises and depresses further transcription of the Clk gene by binding to its promoter. In contrast, PDP 1 activates the Clk gene transcription in the midnight, since PDP 1 peaks 3--4 h later in the night than VRI. Therefore, in the midnight the Clk gene transcription proceeds and by early in the day the Clk RNA peaks in the cytoplasm. During the daytime, VRI and PDP 1 levels are low in the cytoplasm, and at that time the CLK level is high in the cytoplasm, leading to heterodimer formation with CYC (CLK:CYC). This heterodimer moves into the nucleus and binds to vri and Pdp 1 resulting once again in their transcriptional activation.
However, binding of PER:TIM to CLK:CYC in the nucleus, stops the transcriptional activation of vri and Pdp 1 similar to per and tim. Thus, the CLK interconnects both the back loops. (For more clarity, the events in the nucleus were split for each feed--back loop).
By midnight, PER, TIM and DBT enter the nucleus perhaps as a complex (Curtin et al., 1995; Kloss et al., 2001). The phosphorylation of PER and TIM continues in the nucleus by the respective kinases, DBT and SGG (Edery et al., 1994; Zeng et al., 1996; Price et al., 1998; Martinek et al., 2001). Thus, it was implicated that SGG might also be accompanying the PER, TIM and DBT complex. This complex likely binds to the CLK:CYC dimer and stops the transcription of per and tim.
To reinitiate per and tim transcription by CLK:CYC the repression by PER:TIM on CLK:CYC must be relieved. Therefore, the PER:TIM heterodimer must be either detached from CLK:CYC or degraded. Upon light exposure, TIM is degraded and eases the DBT mediated phosphorylation of PER, which is subsequently degraded. In the PER:TIM complex PER is stable, therefore it is suggested that
TIM also inhibits nuclear function of DBT (Rothenfluh et al., 2000). Therefore, ultimately, TIM dissociation (and degradation) from the PER:TIM repressor complex defines the period length of a molecular cycle. SGG phosphorylates TIM, which promotes the latter’s transport from the cytoplasm to the nucleus (Martinek et al., 2001). Similarly, SGG mediated phosphorylation of TIM in the nucleus might trigger the latter’s dissociation from the PER:TIM complex. The dissociation of phosphorylated TIM from PER makes it vulnerable for rapid degradation by light signals (Martinek et al., 2001) or even in the absence of light signals; because it was shown that TIM levels fall before lights are ‘on’ in LD cycle (Zeng et al., 1996).
However, PER alone can act as potent repressor in the complete absence of TIM (Rothenfluh et al., 2000). That is why the per and tim transcription remains depressed even after TIM is degraded by light in LD cycles (Zeng et al., 1996; So and Rosbash, 1997).
4.2.3 CLK, VRI and PDP 1 enhances per and tim transcription
CLK is one of the transcriptional activators for per and tim. The Clk gene shows rhythmic expression of both its RNA and protein like per and tim, but the phase of both oscillations is opposite to that of per and tim RNA. This opposite phase suggested the existence of a second feed--back loop. This additional loop, with Clk in the centre, possibly amplifies per and tim transcriptional rhythms by increasing the amplitude. PER and TIM positively affect the Clk RNA and protein levels as per01 and tim01 mutants have low levels of both Clk RNA and protein (Bae et al., 1998; Lee et al., 1998). On the other hand, per01 ClkJrk and per01 cyc01 double mutants showed high levels of Clk, suggesting that CLK and CYC repress the Clk expression but per and tim derepress the negative action of CLK and CYC on Clk expression (Glossop et al., 1999).
The bZIP transcription factors, VRILLE (VRI) and PAR domain protein 1 (PDP 1) are expressed with a similar phase as per and tim, indicating that both are regulated by the same mechanism as per and tim (Fig. 3; Blau and Young, 1999;
Cyran et al., 2003). This suggests that the CLK:CYC dimer that effects per and tim expression may control vri and Pdp 1 expression too. Indeed, the vri and Pdp 1 RNA levels are low in ClkJrk and cyc01 mutants (Blau and Young, 1999; Cyran et al., 2003). Moreover CLK activates vri expression in vitro in an E--box dependent
manner (Blau and Young, 1999). Similar E--boxes are also present in the Pdp 1 promoter (Cyran et al., 2003).
The expression of the transcription factor vri is positively activated by CLK:CYC, and during early night the VRI protein enters the nucleus and inhibits the transcription of Clk. In contrast, PDP 1 activates Clk expression during the late night (Cyran et al., 2003). Thus VRI and PDP 1 have an opposite effect on Clk.
However, the expression of these proteins is temporally displaced by 3--6 h. PDP 1 is produced maximally around midnight and VRI during early night, therefore the PDP 1 eventually causes a rise in production of CLK in the daytime.
vri levels are intermediate in per01 and tim01 mutants (Blau and Young, 1999), probably due to the relaxed transcription of vri by CLK:CYC, resulting in building up of VRI, which would explain the low levels of Clk RNA in per01 and tim01 mutants (Bae et al., 1998; Lee et al., 1998). An overexperssion of vri results in a reduction of per and tim RNA levels (Blau and Young, 1999), possibly by reducing CLK, which in turn down regulates per and tim expression. The two described feed--back loops are interconnected via CLK. CLK acts as a pivot as follows:
binding of PER:TIM to CLK:CYC depresses the activation of per and tim and simultaneously releases the CLK:CYC--dependent repression of Clk transcription mediated by VRI (Fig. 3).