vrille, Pdp1, and dClock Form a Second Feedback Loop in the Drosophila Circadian Clock

Abstract

The Drosophila circadian clock consists of two interlocked transcriptional feedback loops. In one loop, dCLOCK/CYCLE activates period expression, and PERIOD protein then inhibits dCLOCK/CYCLE activity. dClock is also rhythmically transcribed, but its regulators are unknown. vrille (vri) and Par Domain Protein 1 (Pdp1) encode related transcription factors whose expression is directly activated by dCLOCK/CYCLE. We show here that VRI and PDP1 proteins feed back and directly regulate dClock expression. Repression of dClock by VRI is separated from activation by PDP1 since VRI levels peak 3-6 hours before PDP1. Rhythmic vri transcription is required for molecular rhythms, and here we show that the clock stops in a Pdp1 null mutant, identifying Pdp1 as an essential clock gene. Thus, VRI and PDP1, together with dClock itself, comprise a second feedback loop in the Drosophila clock that gives rhythmic expression of dClock, and probably of other genes, to generate accurate circadian rhythms.

Clk post-transcriptional control denoises circadian transcription both temporally and spatially

“…The latter is constitutively expressed, whereas CLK is present in limiting amounts 4,10 . The CLK-CYC heterodimer also activates the expression of VRILLE (VRI) and PAR-DOMAIN-PROTEIN 1 (PDP1), which have been postulated to be responsible for the oscillation of Clk mRNA 11,12 . Although the role of VRI in repressing Clk transcription is well established, recent reports suggest that PDP1 might not directly regulate Clk 13 .…”

“…The current model states that most (if not all) of this control is through post-translational regulation such as phosphorylation, ubiquitination and other types of modifications [21][22][23][24][25] . Indeed, although Clk mRNA levels display strong transcriptional oscillations 11,12 , CLK protein levels are nearly constant though the day 26 . Moreover, expression of CLK under the tim or per promoter in ClkARK fly strains (in which transcription has the opposite daily phase relative to that under control of the Clk promoter 21 ) does not disrupt circadian behaviour, indicating that flies can adapt or compensate for high levels of CLK 21 .…”

Modelling of circadian rhythms in Drosophila incorporating the interlocked PER/TIM and VRI/PDP1 feedback loops

“…Transcriptional regulation underlying the circadian clock in Drosophila includes two interacting feedback loops, as shown in Figure 1 (Glossop, Lyons et al 1999;Cyran, Buchsbaum et al 2003;Hardin 2005 (Allada, 1998). After initial activation of per and tim expression, there is a 4 h -6 h delay between the peak concentrations of per and tim mRNAs and that of PER and TIM proteins (Zerr, Hall et al 1990;Zeng, Qian et al 1996).…”

Even a stopped clock tells the right time twice a day: circadian timekeeping in Drosophila

“…Here, CLK/CYC activates transcription of vrille (vri) and PAR-domain protein 1ɛ (Pdp1ɛ), which encode related basic leucine zipper transcription factors [39][40][41]. VRI and PDP1ɛ regulate Clk transcription, with VRI acting as a repressor and PDP1ɛ an activator of transcription, causing Clk RNA levels to cycle in opposite phase to per, tim, vri and Pdp1ɛ [39,40]. vri RNA and protein levels peak ∼3-6 h before those of Pdp1ɛ, presumably underpinning the daily rhythm in Clk expression [40].…”

“…VRI and PDP1ɛ regulate Clk transcription, with VRI acting as a repressor and PDP1ɛ an activator of transcription, causing Clk RNA levels to cycle in opposite phase to per, tim, vri and Pdp1ɛ [39,40]. vri RNA and protein levels peak ∼3-6 h before those of Pdp1ɛ, presumably underpinning the daily rhythm in Clk expression [40].…”