Elucidating the Mechanisms of Lipid Droplet Proteome Regulation using a Functional Genomics Approach

Lipid droplets (LDs) are endoplasmic reticulum (ER)-derived organelles that can be formed in nearly all cell types and are highly conserved, from bacteria to plants and animals. LDs are composed of a hydrophobic core of neutral lipids (e.g. triacylglycerols, TAG, and cholesterol esters, CE) encircled by a phospholipid monolayer decorated with a unique complement of resident proteins. By mediating the dynamic storage and release of the contained lipid reserves, LDs play a crucial role in maintaining cellular energy balance within cells and throughout the body. Disruptions in LD homeostasis are associated with numerous metabolic pathologies, including obesity and insulin resistance, hepatic steatosis (fatty liver), cardiovascular disease, and lipodystrophies./nElucidating the underlying molecular mechanisms of aberrant lipid storage is essential for understanding the pathogenesis of metabolic disorders such as hepatic steatosis. As the LD proteome consists of a wide array of proteins and enzymes involved in lipid metabolism, including many neutral lipid synthesis and hydrolysis enzymes (e.g. ACSL3, DGAT2, and ATGL), LD protein composition plays a crucial role in maintaining cellular lipid homeostasis. The major hepatocyte LD scaffolding protein perilipin-2 (PLIN2) is upregulated in patients with hepatic steatosis and mice with diet-induced fatty liver disease. Interestingly, multiple studies report that PLIN2 knockout mice exhibit reduced liver fat content and are protected against diet-induced obesity and hepatic steatosis, suggesting that modulation of PLIN2 levels could serve as a therapeutic strategy for metabolic disease. Despite the critical need for understanding the dynamic regulation of PLIN2 and the LD proteome, the pathways and mechanisms that control PLIN2 (e.g. PLIN2 expression, LD localization, and removal from LDs and subsequent degradation) are incompletely understood./nIn this dissertation, we employ a functional genomics strategy to uncover genes that regulate PLIN2 and LD abundance. We develop a set of genome-edited PLIN2 reporter cell lines and perform a series of CRISPR-Cas9 loss-of-function screens, generating a comprehensive inventory of genes that influence PLIN2 levels under different metabolic conditions. Moreover, we uncouple their effects on PLIN2 expression and post-translational stability. Identified genetic modifiers include canonical genes that control LD metabolism (e.g. ACSL3, DGAT2, PNPLA2, ABHD5) as well as genes with less characterized roles in PLIN2 and LD regulation, such as ubiquitination machinery (e.g. MARCH6, UBE2J2), transcription regulators (e.g. HNF4A, HDAC3), and mitochondrial pathways (e.g. electron transport chain and mitochondrial fatty acid synthesis). These CRISPR screens, and several published screens that focus on different aspects of lipid metabolism, provide the foundation for CRISPRlipid (http://crisprlipid.org), a versatile, online data commons for lipid-related functional genomics data. Together, these studies uncover new mechanisms of PLIN2 regulation and provide an extensive, phenotype-rich resource for the exploration of LD biology and lipid metabolism.

Advisor: Olzmann, James A