Abstract
BackgroundThe 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.ResultsWe 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.ConclusionOur 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.
Highlights
The c-Myc oncogenic transcription factor heterodimerizes with Max, binds specific DNA sites and regulates transcription
TATA binding protein (TBP) is limiting for GAL4-Myc transactivation To determine whether TBP is limiting, we titrated the GAL4-Myc transactivation assays with increasing amounts of TBP plasmid
The GAL4-Myc chimera has significant transactivation activity under the experimental conditions chosen, addition of increasing amounts of TBP plasmid resulted in a corresponding increase in transactivation of GAL4-luciferase reporter (G5TATALuc) from about 10 fold to over 60 fold with the highest amount of TBP plasmid used (Fig. 1)
Summary
The c-Myc oncogenic transcription factor heterodimerizes with Max, binds specific DNA sites and regulates transcription. The c-myc oncogene is implicated in the genesis of many human cancers and accounts for about 70,000 US cancer deaths annually [1,2,3] This oncogene produces the c-Myc transcription factor, which heterodimerizes with Max via the helix-loop-helix-leucine zipper (HLH-Zip) motif to bind specific target DNA sequences and regulate transcription [4,5,6,7,8]. Deletion of Myc Box II appears to affect the induction of certain endogenous target genes but not others [16,17] These observations suggest that transcriptional regulation by Myc is likely to be manifold, involving chromatin modulation as well as direct interaction with components of the basal transcriptional machinery [18,19,20]. This spectrum of activities allows Myc to regulate subsets of genes that are more tightly controlled and susceptible to chromatin modulation, whereas other genes, such as the so-called "housekeeping" genes, may already exist in open chromatin configuration and may be regulated through recruitment of the basal transcriptional machinery
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