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초파리 생체시계에서 dCLOCK 단백질의 조절에 대한 메커니즘과 기능 연구

Molecular mechanism and roles of dCLOCK regulation in Drosophila circadian clock

초록/요약

Circadian clocks adapt to daily environmental changes and produce physiological and behavioral rhythms of approximately 24 h in most living organisms. The common molecular mechanism is the involvement of transcriptional/translational feedback loops (TTFL), which are well conserved in various organisms. The rate-limiting component, CLOCK, is a core transcription factor and essential for the rhythmic expression of the core clock genes. In Drosophila, CLOCK (dCLK) forms a heterodimer with CYCLE (CYC) and transcribes period (per) and timeless (tim) genes and other core clock genes. Newly translated PERIOD (PER) proteins translocate into the nucleus and physically interact with the dCLK/CYC complex to inhibit the transcriptional activity of dCLK/CYC, forming a negative feedback loop. Thus, the activation and repression of CLOCK transcriptional activity by rhythmic interaction among activators and repressors is central to the maintenance of circadian rhythms. In addition to transcriptional regulation, posttranslational regulation, mainly phosphorylation of clock proteins, also plays a crucial role in fine-tuning the 24-h cycle of molecular oscillators. dCLK undergoes daily changes in phosphorylation states; for example, hypo-to-medium phosphorylated isoforms present during mid-day/early night, and medium-to-hyper phosphorylated isoforms present during late night/early day. Mostly, “medium” phosphorylated isoforms of dCLK are observed throughout the day. Previous reports showed that hyper-phosphorylated dCLK by DOUBLETIME (DBT), a homolog of casein kinase Iε, promotes the degradation of dCLK. However, the function of ”medium” phosphorylation of dCLK is unclear at the organismal level. Therefore, a key understanding of how CLOCK regulates the molecular oscillator is important in the circadian pacemaker. To further understand the CLOCK-controlled mechanism in Drosophila, I researched three main aspects in this study. First, to understand how dCLK regulates transcriptional activity, based on the concept that PER physically interacts with dCLK to repress dCLK activity, I initially sought to identify the region in dCLK required for the interaction with PER, a core repressor. I used co-immunoprecipitation with internal deletion dCLK variants and PER in Drosophila S2 cell lines. I identified that amino acids (AA) 657-707 of dCLK, a mouse CLK exon 19 homologous region, which is deleted in Clk/Clk mutant mice, are required for PER interaction in both S2 and HEK293 cells; however, it is not a direct PER-binding domain. dCLK internally deleted from AA657-707, named dCLK-, has attenuated E-box-dependent transcriptional activity in S2 cells. Consistent with this, in transgenic flies expressing dCLK lacking AA657-707 in the Clk null genetic background (termed p{dClk-};Clkout), the peak level and amplitude of core clock gene mRNA rhythms were lower than those in control flies. Behaviorally, p{dClk-};Clkout flies manifested arrhythmic locomotor behavior with no anticipatory activities during photic entrainment. At the molecular level in clock cells, an unexpected pacemaker neuron-specific disturbance of the molecular clockwork was observed. The amplitude of dCLK target proteins significantly dampened in photosensitive neurons, such as ventral lateral neurons (LNvs), while the dCLK target protein cycling was robust in dorsal lateral neurons (LNds) and dorsal neurons (DNs) under light entrainment condition. The alterations of molecular oscillators in LNvs might explain the arrhythmic locomotor behavior rhythms during photic entrainment. Therefore, dCLK AA657-707 plays an important role in both PER interaction and transcriptional activity and is necessary for LNvs-specific transcriptional activation, thereby, regulating photic induced behavior activity. Second, to understand the global physiological roles of dCLK phosphorylation at the organismal level, I initially identified multiple phosphorylation sites on dCLK from Drosophila S2 culture cells by using mass spectrometry. The results in S2 cells showed that global phosphorylation of dCLK decreases its stability and transactivation potential. Transgenic flies expressing the dCLK mutant version, wherein 15 identified serine residues switched to alanine in the Clk null genetic background (termed p{dClk-15A};Clkout), show behavioral rhythms with a 1.5-h shorter period during photic entrainment. In flies, dCLK-15A protein is hypo-phosphorylated and exclusively abundant, which could explain the faster pace of the clock. The daily peak levels in per/tim mRNA and protein reached higher values in dCLK-15A mutant flies, further supporting the notion that dCLK levels are rate-limiting in the clock mechanism. Consistently, the amplitude of dCLK target protein in pacemaker neurons increased with an advanced phase in photic entrainment condition, and it was highly correlated with a shorter period. Therefore, global phosphorylation is essential for dCLK stability, and transactivation potential, ultimately, plays a crucial role in setting the pace of the clock. Finally, from the surprising results that p{dClk-};Clkout flies showed robust oscillation in temperature-sensitive neurons, such as in DNs and not in LNvs, although the flies have low dCLK-mediated transcription activity, I focused on dCLK regulation in temperature entrainment condition. In p{dClk-};Clkout flies, anticipatory activities before temperature transition were observed in temperature cycles, unlikely in light/dark cycles. Importantly, although the amplitude of molecular oscillator in LNvs greatly reduced, the amplitude of molecular rhythms in temperature-sensitive neurons, such as DNs, was robust with a delayed phase, which is correlated with delayed anticipatory activity in temperature cycles. Thus, p{dClk-};Clkout flies manifest improved locomotor activity in temperature entrainment condition, which is likely due to robust molecular oscillations in temperature-sensitive neurons such as DNs. On the contrary, in p{dClk-15A};Clkout flies, the daily activity rhythms failed to maintain synchronization to temperature cycles under constant darkness, although there was no observable impairment in aligning to light/dark cycles. More importantly, pacemaker neuron-specific disturbance of the molecular clockwork was also observed in p{dClk-15A};Clkout flies. The amplitude of dCLK target proteins significantly dampened specifically in DNs, but not in LNvs and LNds, in temperature cycles. Therefore, blocking multiple phosphorylations of dCLK or high levels of dCLK-induced DN-specific disturbance of the molecular clockwork leads to impaired temperature-induced behavior in locomotor activity. Taken together, the results from two different CLOCK mutants suggest that the dCLK/CYC-dependent machinery operates differentially in a subset of pacemaker neurons, which may contribute to specific roles, such as differential sensitivity to entraining cues. Further studies will be required to understand the molecular mechanisms of dCLK regulation in temperature condition further and explore how dCLK/CYC-dependent machinery works differently in cell type- or tissue-specific manner. In conclusion, first, dCLK AA657-707, a mouse dCLK exon 19 homologous region, is required for PER interaction and transactivation potential. It also contributes to photic induced behavioral locomotor rhythms. Second, global phosphorylation of dCLK is crucial for maintaining dCLK abundance properly, playing a critical role in setting the pace of the clock. Finally, pacemaker neuron-specific alterations by two different dCLK mutants suggests that dCLK regulation plays a crucial role in governing circadian entrainment by adjusting the circadian gene expression in a pacemaker neuron-dependent manner.

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목차

I. INTRODUCTION 1
A.Circadian timing system 1
B. The molecular basis of the circadian system 2
C. The regulation of CLOCK activity 4
D. The phosphorylation of clock proteins 7
E. The phosphorylation of CLOCK 9
F. Drosophila Circadian Pacemaker neurons 10
G. Light induced entrainment 11
H. Temperautre induced entrainment 12
I. Aims 13
II. Materials and methods 16
A. Plasmids 16
B. Identification of dCLK phosphorylation sites by mass spectrometry 17
C. Fly strains and transgenic flies 18
D. Behavioral assays 20
E. Luciferase assay 21
F. Quantitative real-time PCR 22
G. Immunoblotting and immunoprecipitation 22
H. Immunohistochemistry and confocal image analysis 23
I. In vitro binding assay 24
III. RESULTS 26
PART A. Roles of amino acids 657-707 of dCLOCK in Drosophila circadian clock 26
1. Amino acids 657-707 of dCLOCK is crucial for PER interaction and transcription activity in S2 cells 26
2. Flies harboring dCLK-Δ manifest arrhythmic behavior in photic entrainment condition 33
3. dCLK-Δ protein is hypo-phosphorylation and stable 37
4. dCLK-Δ impaired binding to PER and TIM in flies 40
5. Molecular rhythms are disrupted in p{dClk-Δ};Clkout flies 42
6. Levels of dCLK target clock proteins are decreased in LNvs of p{dClk-Δ};Clkout flies 46
PART B. Roles of global phosphorylation of dCLOCK in Drosophila circadian clock 56
1. Identification of phosphorylation sites on dCLK in S2 using mass spectrometry 55
2. Flies harboring dCLK-15A display behavioral rhythms with short periods 62
3. dCLK-15A protein is hypo-phosphorylation and stable 67
4. Molecular rhythms are increased in p{dClk-15A};Clkout flies in LD 69
PART C. Mechanism of dCLOCK regulation in Temperature cycles using two different dCLOCK mutants 75
1. p{dClk-Δ};Clkout flies improve anticipatory activity and rhythmicity under TC 75
2. Molecular rhythms in pacemaker neurons of p{dClk-Δ};Clkout flies under TC 78
3. Locomotor behavioral rhythms of p{dClk-Δ};Clkout flies under TC 81
4. Flies harboring dCLK-15A impair synchronization to temperature cycle 88
5. Molecular rhythms are disrupted in p{dClk-15A};Clkout flies after prolong exposure to TC 97
6. Rhythms of dCLK target clock proteins are disrupted in DNs of p{dClk-15A};Clkout flies under TC 100
IV. DISCUSSION 107
PART A. Roles of amino acids 657-707 of dCLOCK in Drosophila circadian clock 107
PART B. Roles of global phosphorylation of dCLOCK in Drosophila circadian clock 112
PART C. Mechanism of dCLOCK regulation in Temperature cycles using two different dCLOCK mutants 118
V.SUMMARY AND CONCLUSION 125
REFERENCES 127
국문요약 139

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