Transcriptional Activity of FOXO Transcription Factors Measured by Luciferase Assays.

Abstract

The Forkhead box O (FOXO) family of transcription factors translates environmental cues into gene expression. FOXO factors are crucial for the maintenance of cell homeostasis, with important roles in cell fate decisions and differentiation. Identification of FOXO target genes requires strict validation by several methods. Luciferase-based reporters are a valuable starting point for determining the transcription-promoting capacity of potential FOXO-binding sites in candidate genes. Luciferase, an enzyme found in bioluminescent organisms catalyzes oxidation of luciferin to produce oxyluciferin together with light, which can be easily detected and measured with a luminometer. Due to their many advantages, transcriptional assays based on luciferase activity are widely used; they are easy, highly reproducible, and very sensitive. Continued improvements in luciferase-based vectors and measurement reagents confer considerable versatility. Luciferase-based reporters are also a reliable approach in the search for unknown components in the signaling pathways that control FOXO factor activity.We previously reported that FOXO transcription factors control expression of the enzyme diacylglycerol kinase α (DGKα) in T cells. DGKα consumes diacylglycerol, a lipid that activates several mitogenic pathways. Here, we describe the use of a luciferase-based promoter bearing the FOXO-binding sites of the DGKα gene to explore the relationship between the expression of this enzyme and stress conditions in NIH3T3 mouse fibroblasts. Our data support a role for FOXO factors in promoting high DGKα levels in conditions of growth factor deprivation. DGKα regulation by FOXO factors correlates with the reported alterations in DGKα expression during cell transformation and cancer progression.