Functional investigation of PERIOD in the mammalian circadian clock

Circadian rhythms are cell-autonomous biological oscillations with a period of approximately 24 hours. They are observed across all kingdoms of life and allow the organisation of physiology to anticipate and accommodate the different environmental and energetic demands of day and night. In human cells, circadian temporal programs are thought to be facilitated by a feedback loop in which PER and CRY proteins form complexes that repress CLOCK·BMAL1-driven transcription of the Period (Per) and Cryptochrome (Cry) genes. Whilst Per clock genes are essential for circadian rhythmicity, little is known about the cell-autonomous mechanisms that confer 24h organisation to the activity and subcellular localisation of PER-containing complexes. In this study, I aimed to provide a molecular understanding for the rhythmic functions of PER2 in human cells, by first assessing the roles of rhythmic Per transcription and rhythmic PER protein abundance in driving circadian rhythmicity. Finally, I aimed to explore how the rhythmic activity of PER helps drives circadian rhythmicity. The contribution of rhythmic Per transcription to circadian rhythmicity was investigated through the use of Per1/2 double knockout cells. The role of rhythmic PER protein abundance was then investigated through a series of constitutive overexpression and proteolysis targeting chimera (PROTAC) acute knockdowns. Next, PER2 activity changes over the cellular circadian cycle were assessed through time-resolved mass spectrometry of the PER2 interactome. Finally, CRISPR-mediated gene editing was used to investigate the circadian subcellular localisation of PER2-containing complexes. I found that neither rhythmic transcription of Per genes, nor rhythmic PER protein abundance is required for circadian rhythms in PER function, and gained novel insight into how PER activity rhythms manifest themselves in human cells. Several novel PER2 interactors were identified and validated, with clear enrichment for RNA-binding proteins, spliceosomal and ribosomal proteins. Several sites of rhythmic phosphorylation were also identified on PER2. Finally, the interaction between PER2 and mTORC1 was characterised in cells and in vitro. Taken together, this work highlights that daily changes in Per transcript abundance or corresponding protein level are insufficient to account for its daily rhythms in function and proposes refinements to the current model that emphasise a central role for PER activity rather than abundance.

Name dc:type.qualificationname

Doctor of Philosophy (PhD)

Level dc:type.qualificationlevel

Doctoral

Grantor dc:publisher.institution

University of Cambridge

Year dc:date.issued

2023

Author dc:creator

• Mihut, Andrei

Advisor dc:contributor.advisor

• O'Neill, John

• PERIOD
• Circadian Rhythm
• Circadian Clock
• Circadian Cell Biology

Licence

• https://apollo8-f-pro.lib.cam.ac.uk/bitstreams/52558a09-f839-44af-8aff-e7539a15cd79/download
• http://purl.org/NET/rdflicense/allrightsreserved

Language dc:language

eng

DOI dc:identifier.doi

https://doi.org/10.17863/CAM.116005

OAI identifier oai:identifier

oai:www.repository.cam.ac.uk:1810/380133