Discovering Transcription Factors and Upstream Regulators of mammalian SIRT3 via Luciferase Reporter Assay

Activation of mammalian SIRT3, a mitochondrial deacetylase, decreases cellular damage in cells by activating an antioxidant enzyme, SOD2, which scavenges Reactive Oxidative Species (ROS), one of the causes of oxidative damage to key biological molecules like DNA. Hence SIRT3 is thought to be involved in regulating cellular aging. Finding regulators of SIRT3 can lead to the development of protocols for reducing oxidative damage levels in cells and ultimately in organisms. The transcripitional co-activator PGC1-alpha is a known activator of SIRT3 but other regulators need to be explored if we want to further understand and control cellular longevity through diverse biological pathways. To achieve this we use a luciferase reporter assay in mouse tissue cells to test how transcription factor genes in our plasmid library can affect SIRT3 expression. Our reporter construct contains the SIRT3 promoter and luciferase downstream in a plasmid that will be transfected into all cells. If a gene from our library regulates SIRT3 either positively or negatively we should see an increase or decrease in the amount of luciferase expressed which will be measured by average luciferase fluorescence intensity per cell via flow cytometry. Cells with PGC1-alpha will be positive controls while cells with only the reporter will be negative controls. When we begin to get results those candidate genes will need to be further identified as either coactivators, transcription factors, or repressors and then study exogenous ways to activate activators or repress repressors to hopefully increase cellular longevity