Investigation into the molecular mechanisms underlying circadian rhythm disruption and human cancer

Human Period2 (PER2) is one of the core mammalian clock factors known to be rhythmically expressed throughout the body and directly binds to the tumor suppressor p53 and its negative regulator, the oncogenic mouse double minute-2 (MDM2) protein. PER2 modulates p53 transcriptional activity by preventing MDM2-mediated ubiquitination and subsequent proteasomal degradation, promoting p53 stability. Critically, one p53 mutation, Y220C (YC), is a hotspot in cancer and uniquely lies at the binding site of PER2 within p53, distant from the interface binding to DNA. We hypothesized that an altered interaction between PER2 and p53YC results in less available PER2 and p53 to function within their canonical pathways by preventing dissociation. We determined that there is stronger association between PER2 and p53YC compared to p53WT and used molecular dynamics simulations to reveal that this altered interaction is stabilized by hydrophobic interactions. These simulations also revealed a potential "wrapping" effect in which PER2 can potentially prevent p53YC from interacting with DNA or tetramerizing with other p53YC monomers, which was supported by experimental evidence from electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) experiments. At the cellular level, Trp53YC luciferase reporter cells were found to have dampened circadian oscillations, altered rhythmicity of core clock gene transcription, and exhibited increased proliferative and survival ability relative to Trp53WT. RNA-seq analysis revealed that PER2 provides a fitness advantage to cells with p53YC gain-of-function activity by modulating the regulation of cellular metabolism, division, and proliferation, promoting an oncogenic phenotype. Together, these findings provide a potential mechanistic link between p53 dysregulation, circadian deregulation, and cancer.

Name thesis:degree_name

Doctor of Philosophy

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Biological Sciences

Department dc:contributor.department

Biological Sciences

Grantor dc:publisher

Virginia Tech

Year dc:date.issued

2023

Author dc:creator

• Janoski, Jesse Ryan

Chair dc:contributor.committeechair

• Finkielstein, Carla V.

Committee members dc:contributor.committeemember

• Smyth, James
• Lemkul, Justin Alan
• Kojima, Shihoko
• Pan, Yuchin Albert

• circadian rhythm
• circadian clock
• Period2
• p53
• hotspot mutation
• cancer

Statement dc:rights

• Creative Commons Attribution-NonCommercial 4.0 International

Licence dc:rights.uri

• http://creativecommons.org/licenses/by-nc/4.0/

Language dc:language.iso

en

Dc Identifier Other

vt_gsexam:38398

OAI identifier oai:identifier

oai:vtechworks.lib.vt.edu:10919/116087