A novel tetracycline-dependent transactivator with E2F4 transcriptional activation domain

Abstract

A tetracycline-controlled gene expression system provides a powerful tool to dissect the functions of gene products. However, it often appears difficult to establish cell lines or transgenic animals stably expressing tetracycline-dependent transactivators, possibly as a result of toxicity of the transactivator domains used. In order to overcome this problem, we developed a novel tetracycline-dependent transactivator that works efficiently in mammalian cells. This transactivator is a fusion of the tet reverse repressor mutant and the transcriptional activating domain of human E2F4, which is ubiquitously expressed in vivo. We demonstrate here that this tetracycline-regulated gene expression system provides a two log transcriptional activation in mammalian cells as assessed by northern blot and luciferase analyses. Combining this system with green fluorescent protein reporter systems or microarray gene expression profiling will facilitate the study of gene function.

Figure 1

The structure of the novel transactivator. The transactivator domain (amino acids 297–413) of E2F4 was fused to reverse type tetracycline repressor. In order to prevent the RB family proteins from binding to E2F4, four residues (amino acids 404–407) of the pocket protein binding domain of E2F4 were deleted. This transactivator is driven by the CMV promoter (prTE4d38).

Figure 2

Kinetics of luciferase activity in ST7L-8 cells by doxycycline. ST7L-8 cells were plated in 24-well plates (at a density of 3 × 10⁴ cells per well). After addition of doxycycline (final concentration 1 µg/ml), luciferase activity was measured at different time points (closed circles). After replacement to tetracycline-free medium, luciferase activity was measured at different time points (open circles).

Figure 3

Northern blot analysis of doxycycline-dependent mtp53–GFP expression in D28 cells. Total RNA (10 µg) was loaded into each lane. The blot was hybridized with a DNA probe for the GFP gene and, as a control for loading, with a GAPDH probe. (A) Kinetics of mtp53–GFP induction in D28 cell. After administration of doxycycline (final concentration 1 µg/ml), cells were harvested at different time points (0, 0.5, 1, 3, 6, 9, 12, 18, 24, 36 and 48 h). (B) Dose–response analysis of doxycycline on the D28 clone. Cells were harvested after 24 h in the presence of doxycycline.

Figure 4

Relative activity of induced gene expression. The expression of mtp53–GFP was standardized with GAPDH. (A) Kinetics of mtp53–GFP induction in D28 cell after administration of doxycycline. (B) Dose–response analysis of doxycycline on the D28 clone.