Cell-type specificity of short-range transcriptional repressors

Abstract

Transcriptional repressors can be classified as short- or long-range, according to their range of activity. Functional analysis of identified short-range repressors has been carried out largely in transgenic Drosophila, but it is not known whether general properties of short-range repressors are evident in other types of assays. To study short-range transcriptional repressors in cultured cells, we created chimeric tetracycline repressors based on Drosophila transcriptional repressors Giant, Drosophila C-terminal-binding protein (dCtBP), and Knirps. We find that Giant and dCtBP are efficient repressors in Drosophila and mammalian cells, whereas Knirps is active only in insect cells. The restricted activity of Knirps, in contrast to that of Giant, suggests that not all short-range repressors possess identical activities, consistent with recent findings showing that short-range repressors act through multiple pathways. The mammalian repressor Kid is more effective than either Giant or dCtBP in mammalian cells but is inactive in Drosophila cells. These results indicate that species-specific factors are important for the function of the Knirps and Kid repressors. Giant and dCtBP repress reporter genes in a variety of contexts, including genes that were introduced by transient transfection, carried on episomal elements, or stably integrated. This broad activity indicates that the context of the target gene is not critical for the ability of short-range repressors to block transcription, in contrast to other repressors that act only on stably integrated genes.

Figure 1

Tet chimeric proteins and reporter constructs. (A) Tet-VP16 consists of TetR fused to the transactivator domain of the herpes simplex virus VP16 (amino acids 363–490). Tet-Stop contains only TetR protein. Tet-repressors contain residues from Knirps (amino acids 80–429), Giant (amino acids 2–389), dCtBP (amino acids 5–353), or Kid (amino acids 12–74). (B) Reporters. TRE-Luc contains seven TREs upstream of the human CMV promoter sequences. cmvTRE-Luc contains the complete CMV enhancer region (−675 to −84) 5′ of the TRE sequences.

Figure 2

Expression of Tet-repressor proteins. HeLa cells were transfected with indicated constructs and nuclear extracts analyzed by gel shift assay. Arrows indicate specific DNA and Tet chimeric protein complexes; arrowhead indicates a nonspecific band present in all samples. Specificity of binding was tested by addition of 10- or 100-fold molar excess unlabeled oligonucleotide. As quantitated by PhosphorImager analysis, Tet-Giant levels were >3-fold higher than those of Tet-dCtBP and Tet-Stop, and Tet-Knirps levels were ≈3-fold higher than those of Tet-Giant. Similar results were obtained in three independent transfection experiments.

Figure 3

Tet-repressor proteins repress basal TRE-Luc promoter in mammalian cells. HeLa and INS-1 cells were transfected with 2 μg of TRE-Luc reporter plasmid and 1 μg of a Tet chimeric repressor construct. Cells were cultured with or without doxycycline (±Dox) before luciferase activity measurement. Tet-Kid was not tested in INS-1 cells. Data are the means ± SD for three independent experiments, each with duplicate measurements. Luciferase activity in the presence of the doxycycline was set at 100.

Figure 4

Dose-dependent repression activity of Tet-repressors in the presence of the Tet-VP16. Two micrograms of TRE-Luc reporter, 1 μg of Tet-VP16 activator, and increasing amounts of Tet-repressor plasmid (0.1, 0.2, 0.5, 1, and 2 μg) were transfected into HeLa (A) and INS-1 (B) cells. Forty hours later, cells were harvested and analyzed for luciferase activity. Data are presented as the means ± SD for three or more independent experiments. In INS-1 cells, 0.5, 1, and 2 μg of Tet-Knirps were used in a single experiment. Luciferase activities are relative to that of Tet-VP16 alone, which was set at 100.

Figure 5

Repression of CMV enhancer activity in transiently transfected HeLa cells. (A) Repression of CMV enhancer activity alone. Cells were transfected with 2 μg of cmvTRE-Luc and 1 μg of Tet-chimera constructs and cultured with or without doxycycline (±Dox). Data represent means ± SD for four or more independent experiments. Luciferase activity in the presence of doxycycline was set at 100. (B) Repression of CMV reporter in the presence of the Tet-VP16 activator. Two micrograms of cmvTRE-Luc, 1 μg of Tet-VP16, and 0.1, 0.2, 0.5, 1, or 2 μg of Tet-repressors were transfected. Data are the means ± SD for three independent experiments with duplicates with luciferase activity set at 100 for the value of Tet-VP16 alone.

Figure 6

Repression of activated gene expression in stably transformed cells. (A) HeLa pCEPTetPLuc and (B) HeLa X1/6 cells containing integrated TRE-Luc reporter genes were transfected with 1 μg of Tet-VP16 and 0.1, 0.2, 0.5, 1, or 2 μg of Tet-repressors. Data are means ± SD for three or more independent experiments. Tet-Knirps was tested in HeLa pCEPTetpLuc cells at the highest levels (1 and 2 μg) in two independent experiments. Luciferase activities are relative to that of Tet-VP16 alone (100).

Figure 7

Cell specificity of Tet-repressors measured in Drosophila S2 cells. TRE-Luc, Tet-VP16, and 0.25, 0.5, 1, or 2 μg of Tet-repressors were transfected into S2 cells as described. Data are means ± SD for three independent experiments. Luciferase activities are relative to that of VP16 alone (100).