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theses

Circadian (≈24 hr) rhythms in physiology and behavior are driven by endogenous cellular clocks that can be synchronized (entrained) by environmental cues, most notably the daily light-dark and temperature cycles. Circadian timing mechanisms in a wide variety of organisms are based on a small set of species-specific clock genes that participate in negative transcriptional feedback loops intertwined with post-transcriptional and post- translational regulatory schemes that ultimately drive cyclical gene expression. Phosphorylation is the most pervasive post-translational modification of clock proteins and is central to setting the pace of the clock. PERIOD (PER), the main repressor in animal clocks, is progressively phosphorylated during its life cycle, which has potent effects on its stability and nuclear localization. In this thesis I used D. melanogaster as an animal model system and identified a new role for PER phosphorylation in entraining to light-dark and temperature cycles. Prior work showed that the light-mediated degradation of TIMELESS (TIM), a key partner of PER, is critical for photic entrainment. My studies have significantly revised this model with the demonstration that the light-mediated degradation of TIM leads to an increase in the phosphorylation of two nearby sites on PER (S826/828), and that blocking phosphorylation at these sites causes altered entrainment, revealing a surprising new role for PER in circadian responses to environmental cues. In related work I contributed to studies that identified phosphorylation sites on the central circadian transcription factor termed CLOCK (CLK), and showed that these modifications are also involved in entrainment. Together, these studies reveal that phosphorylation of PER and CLK is not only critical for setting the pace of the clock but also its ability to interpret external time cues. In another study I found that the miRNA bantam (ban) regulates Clk through three target sites on its 3’ UTR. Flies harboring mutations in ban target sites on Clk show weaker circadian rhythms and less CLK protein staining specifically in the s-LNvs, the key circadian pacemaker cells. These findings show that ban imparts robustness to circadian rhythms by adjusting CLK levels in master pacemaker neurons, and suggest a non- conventional mode-of-operation for ban on Clk expression.

ETD_5854

Ph.D.

Includes bibliographical references

by Evrim Yildirim

doi:10.7282/T30K2B53

ETD doctoral

The author owns the copyright to this work.