Chromatin regulation by RB-interacting proteins in cellular immune functions
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.