NUCLEAR FACTOR ERYTHROID 2 RELATED FACTOR-1 (NRF1) MEDIATES CELASTROL-INDUCED GENE REGULATION, DEPENDING ON ITS HETERODIMERIC INTERACTIONS

Abstract

Oxidative stress has been recognized as critical in human aging and the progression of many chronic diseases, including cancer. Cells undergo oxidative stress when the overproduction of reactive oxygen species (ROS) within the cell outweighs its antioxidant defenses. As a defense mechanism, a series of cytoprotective genes is initiated and regulated by various transcription factors in order to minimize oxidative damage to the cell. NF-E2-related factor 1 (NRF1) is a Cap'N'Collar (CNC) transcription factor family member that plays a major role in regulating genes involved in defense against cell stress and damage. For example, NRF1 is a vital regulator of antioxidant and proteasome genes to counteract ROS and impaired protein homeostasis. Upon stress caused by impaired protein turnover, NRF1 undergoes endoplasmic reticulum to nuclear translocation and binds antioxidant response elements (ARE) located in close proximity to stress defense genes throughout the genome. The result is altered transcription of the associated gene. While this general concept has been established, the mechanism by which NRF1 is processed, selected for nuclear translocation rather than degradation, and the role of post-translational modifications is not understood. Moreover, previous studies show NRF1 must form a heterodimer with other transcription factors such as small musculoaponeurotic fibrosarcoma oncogene homolog (MAF) proteins to bind AREs and regulate gene transcription. The mechanism underlying NRF1 heterodimer formation and ARE binding and whether distinct heterodimers regulate distinct genes is unclear. I hypothesize NRF1 regulates proteasome and oxidative stress defense via specific heterodimer interactions. In this thesis, I describe our identification of the nutraceutical celastrol as a stimulant for NRF1’s transcriptional activity. For centuries, celastrol has been used to treat inflammatory and chronic diseases and more recently found to influence a multitude of stress pathways and suppress chymotrypsin-like activity of the proteasome. I then proceeded to use celastrol to investigate my hypothesis. Using cultured Hep3B cells, I show celastrol elicits dose-dependent inhibition of the proteasome and this increases the level of a cleaved NRF1 protein product known to regulate transcription. Using clustered regularly interspaced short palindromic repeats (CRISPR)/cas9 based technology to produce NRF1 loss-of-function cells, I show celastrol induces transcription of proteasome and oxidative stress defense genes in an NRF1-dependent manner. Likewise, quantitative polymerase chain reaction measurement of immunoprecipitated chromatin revealed that NRF1 binds to AREs in stress defense genes GCLC, GCLM, HO1, NQO1A in a manner that corresponds with transcription regulatory profiles. Moreover, ablation of NRF1 heterodimers MAFG, MAFK, or MAFF resulted in refractory responses to celastrol that partially and nonredundantly matched NRF1 deficiency. Collectively, results of my research support a model whereby NRF1 heterodimerization with MAFK regulates one gene sub-set or program, whereas heterodimerization with MAFG or MAFF regulates another one. In other words, specific heterodimeric interactions coordinately stimulate or repress the transcription of a group of target genes