Cellular clocks on steroids: the mechanism of circadian entrainment by glucocorticoids

Throughout the tree of life most organisms exhibit circadian rhythms to optimize the efficiency of their biological processes and behaviours in light of daily fluctuations in Earth’s environmental conditions. In mammals, glucocorticoid (GC) steroid hormones are vital for signalling time-of-day throughout the body. However, the mechanism by which GCs synchronize and reset the phase of cellular clocks is not completely understood. A plausible model has been proposed, following the classical genomic signalling pathway of its cognate nuclear hormone receptor, the glucocorticoid receptor (GR). The GR activates the transcription of the Period genes which encode the PER proteins, leading to an increase in the abundance of PER. As one of the central components regulating cellular rhythms, PER activity determines the circadian phase at which cells reside. However, much of the evidence for this model is correlatory; uncertainty surrounds which of the PER paralogs are important; and non-transcriptional GR signalling has not been investigated for GC-resetting. In this thesis, I have used contemporary molecular biology methods to directly test the 20-year-old canonical model for GC-resetting and elucidate its mechanistic underpinnings. First, I established a robust cellular assay to characterise GC-resetting in multiple in vitro model systems, demonstrating its potency both in vitro and in vivo. Mechanistic insight into GC-resetting was achieved by taking a GR-centric approach, in which I developed a GR KO-complementation system to investigate which components and functional attributes of the GR are important for its signalling function. I found that nuclear localisation is essential; as well as a functional N-terminal domain, activation function domain 2 region (AF-2), and DNA-binding domain- characteristics suggestive of a transcriptional mechanism. Live-cell microscopy revealed that GR subcellular translocation is highly dynamic, heterogenous between cells and dispensable for GC signalling. Next, using nascent sequencing coupled with mRNA sequencing, I found that, contrary to the proposed model, Per1 is regulated by GCs post-transcriptionally and that GR activation overrides cell-autonomous circadian variation in gene expression at both the nascent RNA and mRNA level. Moreover, chemi-genetic knockdown (KD) of endogenous PER and CRY proteins revealed that PER2 and CRY do not function as a primary GC effectors, whereas PER1 is important for mediating GC-resetting of transcriptional oscillations. Furthermore, quantitative proteomics exposes a clear disconnect between GC-mediated changes observed in transcription and the changes observed at the protein level. Ultimately, based on the evidence presented in this thesis, I propose a more complex cellular model for GC-resetting which involves both transcriptional and post-transcriptional mechanisms as well as revealing the aspects of the GR which are critical to its phase-resetting function.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2025

Author dc:creator

• Edmondson, Anna

Advisor dc:contributor.advisor

• O'Neill, John

• Circadian rhythms
• Cellular biology
• Nuclear hormone receptor signalling

Licence

• https://www.repository.cam.ac.uk/bitstreams/33754758-0279-4a51-96c0-3f01808a6ade/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

Author Identifier

0009-0006-8318-8767

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/392747