title: Luciferase-based reporter to monitor the transcriptional activity of the SIRT3 promoter.
abstract: Sirtuin 3 (SIRT3) is a major regulator of oncometabolism. Indeed, the activity of SIRT3 significantly affects the response to oxidative stress, glycolytic proficiency, and tumorigenic potential of malignant cells. Thus, a system to accurately measure the transcriptional activity of the SIRT3 promoter could facilitate the identification of novel antineoplastic agents or have diagnostic applications. Here, we describe all the steps involved in the development of a luciferase-based reporter system to measure the activation of the human SIRT3 promoter, encompassing the design of appropriate primers, the cloning of the promoter fragment, and its site-directed mutagenesis. We validated this system in human embryonic kidney 293T cells, taking advantage of the renowned ability of the transcription factor estrogen-related receptor α to transactivate SIRT3. Moreover, here we demonstrate that SIRT3 expression is responsive to rapamycin, a small inhibitor of mammalian target of rapamycin that has been extensively employed as a caloric restriction mimetic. Finally, we provide an overview of the complementary molecular biology techniques that might be employed to further verify the reliability of this system.
authors.affiliation: Harvard School of Engineering and Applied Sciences, Cambridge, Massachusetts, USA; Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA.
authors.forename: F Kyle
authors.initials: FK
authors.lastname: Satterstrom
authors.affiliation: Department of Cell Biology, Harvard Medical School, Boston, Massachusetts, USA. Electronic address: marcia_haigis@hms.harvard.edu.
authors.forename: Marcia C
authors.initials: MC
authors.lastname: Haigis
mesh_terms: D001483:Base Sequence; D015153:Blotting, Western; D017931:DNA Primers; D017930:Genes, Reporter; D057809:HEK293 Cells; D006801:Humans; D008156:Luciferases / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D056566:Sirtuin 3 / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D017931:DNA Primers; D008156:Luciferases; C466093:SIRT3 protein, human; D056566:Sirtuin 3
keywords: Caloric restriction mimetic; Luciferase-based reporter plasmid; Oncometabolism; Promoter analysis; Rapamycin; SIRT3