A Genetic and Biochemical Examination of Phosphorylation Regulation of Temperature Compensation and Feedback-Loop Closure of the Circadian Clock

Author

Elizabeth-Lauren Stevenson, Geisel School of MedicineFollow

Author ORCID Identifier

https://orcid.org/0000-0002-1411-7360

Date of Award

3-2026

Document Type

Thesis (Ph.D.)

Department or Program

Molecular and Systems Biology

First Advisor

Jay Dunlap

Abstract

An internal timekeeping mechanism, “circadian rhythms”, has evolved to enable organisms to anticipate daily environmental cycles so they may separate appropriate day/night functions temporally. Circadian rhythms are defined by their ability to entrain to external cues, to continue without such cues, and to maintain the rate of their oscillation despites changes in the environment (“compensation”). The functionally conserved molecular clock in animals and fungi is a transcription-translation feedback loop (TTFL) in which the positive arm of the clock activates transcription of the negative arm of the clock, which in turn feeds back to inhibit the action of the positive arm. There exist many layers of regulation of the core TTFL including regulation by phosphorylation. This thesis investigates the role of phosphorylation regulation in two key aspects of circadian rhythms: temperature compensation and feedback-loop closure.

Previous work has suggested that kinases contribute to temperature compensation (TC) in Neurospora. We dive deeper into this model by using novel alleles of CKII and CKI, Dcka and ck-1a^D135G, to investigate the role that these kinases play in the TC mechanism and the relationship between them. By measuring the phospho-occupancy on FRQ across temperature using mass spectrometry and by performing epistasis experiments with different alleles of CKII and CKI, we determined that CKI is the primary regulator of clock speed (period length) at cold temperatures and CKII is the primary regulator of clock speed at warm temperatures, and that there are distinct regions on FRQ whose phosphorylation contributes to maintaining TC.

We also tested whether period length determination could be uncoupled from negative arm stability in mammals, as has been found in Neurospora where the feedback loop is closed by negative arm hyper-phosphorylation. The ubiquitin ligases b-TrCP1/2 target PER for degradation. We employed a Cre-lox system to induce the removal of b-TrCP2 in a b-TrCP1 null background, and to our surprise, found arrhythmicity in the core clock, unlike in Neurospora. Therefore, feedback-loop closure represents a deviation in TTFL functional conservation, and our results suggest that FRQ may play a functional role more similar to a hybrid of both PER and CRY, rather than just PER.

Recommended Citation

Stevenson, Elizabeth-Lauren, "A Genetic and Biochemical Examination of Phosphorylation Regulation of Temperature Compensation and Feedback-Loop Closure of the Circadian Clock" (2026). Dartmouth College Ph.D Dissertations. 465.
https://digitalcommons.dartmouth.edu/dissertations/465