The construction and analysis of low TCF11/Nrf1 expressing plasmids : regarding transactivational ability and intracellular localization

Abstract

TCF11 is a regulatory transcription factor belonging to the CNC-bZIP family. The specific biological function of this protein is still unknown. However, knockout studies in mice have revealed its importance during embryo development, and other studies have also displayed its involvement in the cell’s defense system against oxidants and carcinogens. The transactivating ability and intracellular localization of TCF11 and the isoform Nrf1 have been studied in cells using high expression plasmids. Due to the recent findings that over-expression of TCF11 in transfected cells caused an increase in cell mortality, the need for lower TCF11/Nrf1 expressing plasmids emerged. An additional reason for constructing the low expression plasmids was to study the localization and transactivating abilities of the proteins at levels closer to the endogenous situation. The transactivating ability was estimated by measuring the luciferase activity in COS-1 cells transiently co-transfected with a reporter plasmid and a high or low TCF11/Nrf1 expressing plasmid. The intracellular localization images were acquired by means of epifluorescence and confocal microscopy. The initial low expression constructs proved unsuitable due to the empty vector’s ability to cause indirect activation of the reporter plasmid. The second set of constructs were low expression plasmids that permitted verification of the nuclear detainment of Nrf1. However, further intracellular compartmentalization could not be detected for either Nrf1 or TCF11. In addition, TCF11 displayed a higher transactivating ability compared to Nrf1.