Circadian Transcription Contributes to Core Period Determination in Drosophila

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Abstract
The Clock–Cycle (CLK–CYC) heterodimer constitutes a key circadian transcription complex in Drosophila. CYC has a DNA-binding domain but lacks an activation domain. Previous experiments also indicate that most of the transcriptional activity of CLK–CYC derives from the glutamine-rich region of its partner CLK. To address the role of transcription in core circadian timekeeping, we have analyzed the effects of a CYC–viral protein 16 (VP16) fusion protein in the Drosophila system. The addition of this potent and well-studied viral transcriptional activator (VP16) to CYC imparts to the CLK–CYC-VP16 complex strongly enhanced transcriptional activity relative to that of CLK–CYC. This increase is manifested in flies expressing CYC-VP16 as well as in S2 cells. These flies also have increased levels of CLK–CYC direct target gene mRNAs as well as a short period, implicating circadian transcription in period determination. A more detailed examination of reporter gene expression in CYC-VP16–expressing flies suggests that the short period is due at least in part to a more rapid transcriptional phase. Importantly, the behavioral effects require a period (per) promoter and are therefore unlikely to be merely a consequence of generally higher PER levels. This indicates that the CLK–CYC-VP16 behavioral effects are a consequence of increased per transcription. All of this also suggests that the timing of transcriptional activation and not the activation itself is the key event responsible for the behavioral effects observed in CYC-VP16-expressing flies. The results taken together indicate that circadian transcription contributes to core circadian function in Drosophila. The existence of circadian clocks, which allow organisms to predict daily changes in their environments, have been recognized for centuries, yet only recently has the molecular machinery responsible for their generation been uncovered. The current model in animals posits that interlocked feedback loops of transcription-translation produce these 24-hour rhythms. In fruit flies, the transcription loop contains a key activator complex, composed of the transcription factors Clock and Cycle. This CLK-CYC complex stimulates the synthesis of repressor proteins like Period and Timeless, which repress the activator complex. The synthesis–repression cycle takes precisely 24 hours under environmental conditions that influence the circadian period. An almost identical process relies on the ortholog proteins CLK-BMAL in mammals. Recent findings have challenged the transcription-translation feedback model and suggest that circadian transcription is an output process and that the post-translational modification of clock proteins is the real central pacemaker mechanism. In the present study, we have manipulated the levels and strength of the CLK-CYC complex. The results demonstrate that its activity is vital for proper period determination and thus indicate that the transcriptional feedback loop is part of the core circadian mechanism.