Phosphorylation of the Transcription Activator CLOCK Regulates Progression through a ∼24-h Feedback Loop to Influence the Circadian Period in Drosophila

Mahesh, Guruswamy; Jeong, Eun; Ng, Fanny S.; Liu, Yixiao; Gunawardhana, Kushan; Houl, Jerry H.; Yildirim, Evrim; Amunugama, Ravi; Jones, Richard C.; Allen, David L.; Edery, Isaac; Kim, Eun Young; Hardin, Paul E.

doi:10.1074/jbc.m114.568493

Cited by 35 publications

(48 citation statements)

References 59 publications

(97 reference statements)

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“…To test this model, we performed ChIP assays using CWO antiserum on wild-type, Clk out and per 01 flies collected at ZT2 and ZT14 in LD. In Clk out flies, which necessarily lack CLK-CYC heterodimers [33], CWO is bound to tim E-boxes at both ZT2 and ZT14 with binding signals comparable to the strong CWO binding in wild-type flies at ZT14 (Fig 5A). In contrast, in per 01 flies, which lack PER-dependent repression of CLK-CYC activation [34], low binding signals of CWO were detected at ZT2 and ZT14, indicating that PER is indeed required for CWO to bind E-boxes (Fig 5A).…”

Section: Resultsmentioning

confidence: 99%

CLOCKWORK ORANGE Enhances PERIOD Mediated Rhythms in Transcriptional Repression by Antagonizing E-box Binding by CLOCK-CYCLE

Zhou

Hardin

2016

PLoS Genet

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The Drosophila circadian oscillator controls daily rhythms in physiology, metabolism and behavior via transcriptional feedback loops. CLOCK-CYCLE (CLK-CYC) heterodimers initiate feedback loop function by binding E-box elements to activate per and tim transcription. PER-TIM heterodimers then accumulate, bind CLK-CYC to inhibit transcription, and are ultimately degraded to enable the next round of transcription. The timing of transcriptional events in this feedback loop coincide with, and are controlled by, rhythms in CLK-CYC binding to E-boxes. PER rhythmically binds CLK-CYC to initiate transcriptional repression, and subsequently promotes the removal of CLK-CYC from E-boxes. However, little is known about the mechanism by which CLK-CYC is removed from DNA. Previous studies demonstrated that the transcription repressor CLOCKWORK ORANGE (CWO) contributes to core feedback loop function by repressing per and tim transcription in cultured S2 cells and in flies. Here we show that CWO rhythmically binds E-boxes upstream of core clock genes in a reciprocal manner to CLK, thereby promoting PER-dependent removal of CLK-CYC from E-boxes, and maintaining repression until PER is degraded and CLK-CYC displaces CWO from E-boxes to initiate transcription. These results suggest a model in which CWO co-represses CLK-CYC transcriptional activity in conjunction with PER by competing for E-box binding once CLK-CYC-PER complexes have formed. Given that CWO orthologs DEC1 and DEC2 also target E-boxes bound by CLOCK-BMAL1, a similar mechanism may operate in the mammalian clock.

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Section: Resultsmentioning

confidence: 99%

CLOCKWORK ORANGE Enhances PERIOD Mediated Rhythms in Transcriptional Repression by Antagonizing E-box Binding by CLOCK-CYCLE

Zhou

Hardin

2016

PLoS Genet

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“…In addition, NEMO over-expression decreases CLK levels and consequently delays PER and TIM accumulation and causes long period. Recently, it was found that CLK is phosphorylated at >15 sites in cell culture [36,37]. Mutation of all of these sites to alanine (CLK-15A) results in hypo-phosphorylated CLK, which has increased stability and transcriptional activity.…”

Section: Post-translational Controlmentioning

confidence: 99%

The molecular ticks of the Drosophila circadian clock

Tataroglu

Emery

2015

Current Opinion in Insect Science

110

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Drosophila is a powerful model to understand the mechanisms underlying circadian rhythms. The Drosophila molecular clock is comprised of transcriptional feedback loops. The expressions of the critical transcriptional activator CLK and its repressors PER and TIM are under tight transcriptional control. However, posttranslational modification of these proteins and regulation of their stability are critical to their function and to the generation of 24-hr period rhythms. We review here recent progress made in our understanding of PER, TIM and CLK posttranslational control. We also review recent studies that are uncovering the importance of novel regulatory mechanisms that affect mRNA stability and translation of circadian pacemaker proteins and their output.

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“…These daily activity patterns were highly similar to the daily activity patterns of Clk out ( Fig. 2D) and Clk Jrk flies, neither of which expresses functional dCLK protein (33)(34)(35). Under constant dark conditions, the p{dClk-Δ},2M;Clk out flies showed dampened morning and evening activity peaks with delayed phases, whereas the p{dClk-Δ},4M;Clk out flies did not manifest evident peaks of activity ( Fig.…”

Section: Results Dclk Internally Deleted For Aa657-707 (Dclk-δ) Showsmentioning

confidence: 48%

Pacemaker-neuron–dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation

Lee

Cho

Kang

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Circadian clocks are composed of transcriptional/translational feedback loops (TTFLs) at the cellular level. In Drosophila TTFLs, the transcription factor dCLOCK (dCLK)/CYCLE (CYC) activates clock target gene expression, which is repressed by the physical interaction with PERIOD (PER). Here, we show that amino acids (AA) 657-707 of dCLK, a region that is homologous to the mouse Clock exon 19-encoded region, is crucial for PER binding and E-box-dependent transactivation in S2 cells. Consistently, in transgenic flies expressing dCLK with an AA657-707 deletion in the Clock (Clk out ) genetic background (p{dClk-Δ};Clk out ), oscillation of core clock genes' mRNAs displayed diminished amplitude compared with control flies, and the highly abundant dCLKΔ657-707 showed significantly decreased binding to PER. Behaviorally, the p{dClk-Δ};Clk out flies exhibited arrhythmic locomotor behavior in the photic entrainment condition but showed anticipatory activities of temperature transition and improved free-running rhythms in the temperature entrainment condition. Surprisingly, p{dClk-Δ};Clk out flies showed pacemakerneuron-dependent alterations in molecular rhythms; the abundance of dCLK target clock proteins was reduced in ventral lateral neurons (LN v s) but not in dorsal neurons (DNs) in both entrainment conditions. In p{dClk-Δ};Clk out flies, however, strong but delayed molecular oscillations in temperature cycle-sensitive pacemaker neurons, such as DN 1 s and DN 2 s, were correlated with delayed anticipatory activities of temperature transition. Taken together, our study reveals that the LN v molecular clockwork is more sensitive than the clockwork of DNs to dysregulation of dCLK by AA657-707 deletion. Therefore, we propose that the dCLK/CYC-controlled TTFL operates differently in subsets of pacemaker neurons, which may contribute to their specific functions.ircadian timing systems are composed of cell-autonomous oscillators that enable living organisms to anticipate environmental cyclic changes, thereby orchestrating behavior and physiology throughout the day. The cell-autonomous oscillator contains transcriptional/translational feedback loops (TTFLs) composed of positive and negative molecular components, driving the rhythmic oscillation of gene expression with 24-h periodicity (1, 2). In Drosophila, the positive components are the basic helix-loop-helixcontaining and Period-Arnt-Sim (PAS)-containing transcription factor dCLOCK (dCLK) and CYCLE (CYC), which form a heterodimer and rhythmically bind to E-box sequences (CACGTC) to activate transcription of clock genes and clock-controlled genes (reviewed in ref. 1). In the core loop of the TTFL, dCLK/CYC transcribes period (per) and timeless (tim) genes and the translated PER and TIM proteins form heterodimers and translocate into the nucleus during mid-evening. The PER/TIM heterodimers then physically interact with the dCLK/CYC complex to inhibit dCLK/CYC-activated transcription. Degradation of PER and TIM proteins by timely controlled posttranslational modifications ul...

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Phosphorylation of the Transcription Activator CLOCK Regulates Progression through a ∼24-h Feedback Loop to Influence the Circadian Period in Drosophila

Cited by 35 publications

References 59 publications

CLOCKWORK ORANGE Enhances PERIOD Mediated Rhythms in Transcriptional Repression by Antagonizing E-box Binding by CLOCK-CYCLE

CLOCKWORK ORANGE Enhances PERIOD Mediated Rhythms in Transcriptional Repression by Antagonizing E-box Binding by CLOCK-CYCLE

The molecular ticks of the Drosophila circadian clock

Pacemaker-neuron–dependent disturbance of the molecular clockwork by a Drosophila CLOCK mutant homologous to the mouse Clock mutation

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