Posttranslational regulation of Drosophila circadian clocks

Circadian clocks enable organisms to anticipate predictable environmental changes over 24-hour day-night cycles on Earth to promote health and fitness. Since the discovery of the first clock gene in Drosophila melanogaster, Drosophila has been a model organism revealing the molecular underpinnings of animal circadian clocks. The first two chapters of this thesis will investigate the regulation of molecular clocks by posttranslational mechanisms. In particular, we will investigate the functions of core clock protein phosphorylation in Drosophila. Despite the importance of rhythmic gene expression programs produced by the molecular clock in generating daily biological rhythms, it has now been established that phosphorylation of core clock proteins that make up the molecular clock represent central and conserved timing mechanisms across organisms. Chapter One will investigate mechanisms by which phosphorylation regulates the major circadian transcriptional activator CLOCK (CLK) of rhythmic genes in D. melanogaster. CLK phosphorylation states exhibit daily rhythms and regulate its abundance, subcellular localization, and transcriptional activity. We characterized the role of casein kinase 1 alpha (CK1a) as a novel CLK kinase. We identified CK1a-dependent CLK phosphorylation sites using mass spectrometry proteomics. We next characterized the function of phosphorylation at Serine 13 (S13) residue. Our results showed that CLK(S13) phosphorylation reduces the binding activity of CLK to circadian promoters, therefore reducing CLK transcriptional activity. We also showed that CK1a-CLK interaction is dependent on PERIOD (PER), the circadian transcriptional repressor that is known to inhibit CLK function. We revealed a mechanism by which repressor-dependent phosphorylation of an activator inhibits its transcriptional activity and thus closing the transcriptional-translational feedback loop (TTFL) of the molecular clock. In Chapter Two, we focused on a comprehensive investigation of phosphorylation on TIMELESS (TIM), the heterodimeric partner of PER. We first identified phosphorylation sites on PER-bound TIM using mass spectrometry. We found that abolishing phosphorylation at some of these residues caused altered circadian behavioral rhythms. Phosphorylation at Serine 1404 (S1404) residue promotes TIM nuclear accumulation by reducing its interaction with exportin 1 (XPO1), a nuclear export machinery. Reduced nuclear localization of TIM in S1404A nonphosphorylatable fly mutants not only resulted in lower level of nuclear PER and TIM, but also showed dampened daily rhythms in CLK phosphorylation. This is likely caused by reduced recruitment of CLK kinase by PER-TIM complex. Taking Chapter One and Two together, we provide mechanistic insights into CLK and TIM phosphorylation and how these posttranslational modifications are indispensable for the maintenance of 24-hour molecular oscillation to regulate circadian rhythms. Chapter Three will review our current understanding of the function of Drosophila TIM (dTIM) and mammalian TIM (mTIM). dTIM is a cardinal clock protein, whereas the role of mTIM in regulating circadian rhythms are under debate since it was first identified over two decades ago. We will summarize the circadian and non-circadian roles of the two TIM paralogs and discuss the potential mechanisms by which mTIM regulates circadian rhythms via its non-circadian roles. We will conclude Chapter Three by summarizing recent findings about potential functional parallel between mTIM and dTIM. Overall, the research included in this thesis will provide mechanistic insights into the regulation of animal circadian clocks and contribute to future development of therapeutics for clock-related human diseases including cancer