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Thesis Format

Integrated Article

Degree

Doctor of Philosophy

Program

Biochemistry

Supervisor

Dick, Frederick A.

Abstract

The retinoblastoma protein (RB) is historically known for its function in cell cycle control. However, mice carrying targeted Rb1 mutations have revealed that RB serves various non-cell cycle control roles. Notably, RB acts as a scaffold that recruits chromatin regulatory proteins, condensin II and enhancer of zeste homolog 2 (EZH2). These complexes protect the genome integrity through maintaining proper chromosome condensation, long range contacts, and transcriptionally repressive histone modification. This thesis explores the mechanistic links that regulate such RB-condensin II complex or that are leveraged upon pharmacological inhibition of the RB-EZH2 complex. First, I identified potential phosphorylation sites in the RB C-terminus (RBC) that may be downstream of T cell receptor (TCR) crosslinking in Jurkat leukemia T cells. I generated and validated phospho-specific antibodies that detect S838/T841 phosphorylation. Upon TCR crosslinking, S838/T841 phosphorylation was indeed detected. p38 mitogen activated protein kinase (MAPK), a known downstream target of TCR signaling, directly phosphorylated RB at S838/T841. Through Jurkat cells overexpressing a phospho-ablated mutant RB construct, I show that S838/T841 phosphorylation is required to dissociate condensin II from chromatin and facilitate chromosome decondensation. In the subsequent chapter, I investigated the consequence and mechanism of pharmacologically inhibiting EZH2. Upon EZH2 inhibition, splenic B cells specifically were eliminated in vivo, which was associated with increased transcription of repetitive elements. Using CRISPR-Cas9 to generate a novel mouse line with abrogated detection of repetitive element transcripts, named RIC mutant, I show that EZH2 inhibition signals inflammatory chemokine expression. This was mediated through reduced histone methylation by EZH2, followed by upregulation of repetitive elements and their detection by three cytosolic pattern recognition receptors (PRRs), RIG-I, MDA5 and cGAS. These PRRs were required to recruit pro-inflammatory immune cells to the spleen in vivo and eliminate B cells. Lastly, I show that RIG-I and cGAS are indispensable for activating anti-viral interferon gene expression in mouse melanoma cells upon EZH2 inhibition. Taken together, this work uncovers regulatory and molecular mechanisms that underlie a biological function or targeted inhibition of chromatin regulatory proteins recruited by RB in immune cells.

Summary for Lay Audience

The retinoblastoma protein (RB) was first discovered as a factor whose loss caused retinoblastoma in children. As such, it became known as a tumour suppressor. Subsequent molecular studies discovered that RB’s tumour suppressive function comes from its ability to control cell cycle, a process where cells divide and proliferate. Indeed, uncontrolled cell cycle can cause cancer. Recently, various studies have found that RB has other roles outside of cell cycle control. RB acts as a scaffold for two proteins called condensin II and EZH2 to contact DNA. Condensin II and EZH2 are proteins that regulate chromatin—a higher order structure of DNA that is compacted and organized in three-dimension. This thesis investigates the mechanisms that govern RB-condensin II interaction, and the effect of inhibiting EZH2 function. First, I discovered that RB is modified at poorly characterized sites by phosphorylation, deposition of negatively charged atoms, upon stimulation that mimics T cell activation in Jurkat leukemia T cells. This activated a series of proteins to directly phosphorylate RB. This event caused both RB and condensin II to be dissociated from chromatin. Consistent with known condensin II function, its loss from chromatin caused chromatin to become less compact. Jurkat cells that express RB that cannot be phosphorylated were resistant to such consequences. Next, EZH2 inhibition eliminated B cells in the spleen, and caused repetitive DNA sequences to be copied into RNA. We created a novel mutant mouse line that is unable to detect such repetitive RNA to test whether it is linked to B cell death. Indeed, these mice did not activate inflammatory immune genes compared to control upon EZH2 inhibition. Importantly, EZH2 inhibition comparably blocked EZH2 function in both WT and mutant mice. The mutant mice were resistant to B cell death and infiltration of inflammatory immune cells in the spleen compared to control. Lastly, I report that detection of repetitive RNA is also important to activate anti-viral signaling upon EZH2 inhibition in cancer cells. Overall, this thesis uncovers a regulatory mechanism for RB-condensin II, and a molecular pathway activated upon inhibition of EZH2 in immune cells.

Creative Commons License

Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License
This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 4.0 License.

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