Stimulation of Myc transactivation by the TATA binding protein in promoter-reporter assays

Abstract

Background: The c-Myc oncogenic transcription factor heterodimerizes with Max, binds specific DNA sites and regulates transcription. The role of Myc in transcriptional activation involves its binding to TRRAP and histone acetylases; however, Myc's ability to activate transcription in transient transfection assays is remarkably weak (2 to 5 fold) when compared to other transcription factors. Since a deletion Myc mutant D106-143 and a substitution mutant W135E that weakly binds TRRAP are still fully active in transient transfection reporter assays and the TATA binding protein (TBP) has been reported to directly bind Myc, we sought to determine the effect of TBP on Myc transactivation.

Results: We report here a potent stimulation of Myc transactivation by TBP, allowing up to 35-fold transactivation of reporter constructs. Although promoters with an initiator (InR) element briskly responded to Myc transactivation, the presence of an InR significantly diminished the response to increasing amounts of TBP. We surmise from these findings that promoters containing both TATA and InR elements may control Myc responsive genes that require brisk increased expression within a narrow window of Myc levels, independent of TBP. In contrast, promoters driven by the TATA element only, may also respond to modulation of TBP activity or levels.

Conclusion: Our observations not only demonstrate that TBP is limiting for Myc transactivation in transient transfection experiments, but they also suggest that the inclusion of TBP in Myc transactivation assays may further improve the characterization of c-Myc target genes.

Figure 1

TBP stimulates GAL4-Myc transactivation. The reporter G5TATALuc contains five GAL4 binding sites followed by a minimal TATA box and the luciferase cDNA. Activator alone: the reporter was cotransfected with GAL4(1-147) DNA binding domain (GALO), GAL4 fused to Myc residues 1-262 (GM1-262), GM1-262 with residues 106–143 deleted (GM1-262D106), or GM1-262 with a substitution of tryptophan 135 to glutamate (GMW135E) alone. TBP at increasing input plasmid amounts (μg indicated on the abscissa) was cotransfected into CHO cells with the reporter alone (TBP) or with the indicated GAL4 plasmids. Bars are shown as averages with standard deviation (n = 10).

Figure 2

TBP does not stimulate initiator driven luciferase reporter G5INRLuc that contains five GAL4 binding sites. Either G5INRLuc or G5TATALuc (see Fig. 1) was cotransfected into CHO cells with GM1-262 alone or with increasing amounts of TBP (μg indicated on the abscissa). For comparison, data points for G5TATALuc are the same as those shown in Fig. 1. Note that TBP does not stimulate GAL4Myc transactivation of G5INRLuc; there is a slight inhibition by TBP. Bars are shown as averages with standard deviation (n = 4).

Figure 3

TBP does not stimulate the activation of G5TATALuc by GAL4USF1. For comparison, the stimulation of G5TATALuc by GAL4Myc (GM1-262) and TBP is shown. By contrast increasing amounts of TBP (μg indicated on the abscissa) does not increase reporter activity that is stimulated by USF TAD. Bars are shown as averages with standard deviation (n = 4). Plasmids were transfected into CHO cells.

Figure 4

Activation of the lactate dehydrogenase A promoter construct (LDHLuc) by Myc is increased by TBP. Activator alone: cotransfection with LDHLuc and MLV-LTR driven Myc expression vectors: MLV, empty expression vector; Myc, wild-type Myc; dHLH, helix-loop-helix deletion mutant D371-412; W135E, substitution mutant with glutamate replacing tryptophan 135. Increasing amounts of TBP (μg indicated on the abscissa) was cotransfected with Myc and reporter constructs. Bars are shown as averages with standard deviation (n = 10). Transfections were performed using NIH 3T3 cells.

Figure 5

Myc DNA binding sites in the LDHA promoter construct (LDHLuc) is required for TBP stimulation of Myc-mediated transactivation. Wild-type LDHLuc or a mutant promoter construct (LDHDMLuc), which has both Myc E-boxes mutated from 5'-CACGTG-3' to 5'-CCCGGG-3', were cotransfected with a constant amount of MLV-LTR driven wild-type c-myc plasmid and increasing amounts of TBP (μg indicated on the abscissa). Bars are shown as averages with standard deviation (n = 6). Transfections were performed using NIH 3T3 cells.

Figure 6

The initiator element in the adenoviral major late promoter luciferase construct (pGLMLP Luc) increases the response to Myc but does not allow further stimulation by TBP. Compared with the reporter containing a mutant InR (pGLMLP dINR Luc), which displays a highly synergistic stimulation by Myc and TBP, the wild-type pGLMLP Luc construct briskly responds to Myc independent of increasing amounts of TBP (μg indicated on the abscissa). Bars are shown as averages with standard deviation (n = 10). Transfections were performed using NIH 3T3 cells.

Figure 7

Activation of the human CDK4 promoter (CDK Luc) by Myc is further stimulated by increasing amounts of TBP (μg indicated on the abscissa). The synergy between Myc and TBP is dependent on the Myc binding sites, which are mutated in the reporter CDK EMut Luc. Bars are shown as averages with standard deviation (n = 6). Transfections were performed using NIH 3T3 cells.

Figure 8

Activation of the ornithine decarboxylase (ODC) intronic E-boxes driven luciferase reporter (ODC Luc) by Myc is further stimulated by TBP (μg indicated on the abscissa). The relative marked stimulation of the ODC sequences by Myc and TBP is compared with the more diminished stimulation of the LDHA promoter (LDH Luc) previously shown in Fig. 4. Bars are shown as averages with standard deviation (n = 10). Transfections were performed using NIH 3T3 cells.

Figure 9

Synergy between Myc and TBP to activate ODC Luc (see Fig. 8) is diminished by mutations in TBP that inhibits TATA box binding (TATA def), interaction with RNA polymerase II (pol2 def) or interaction with RNA polymerase III (pol3 def). Myc (1 μg) was cotransfected with ODC Luc and 4 μg of wild-type or mutant TBP constructs. Bars are shown as averages with standard deviation (n = 4). Transfections were performed using NIH 3T3 cells.