INFLUENCE OF PROMOTER POTENCY ON THE TRANSCRIPTIONAL EFFECTS OF YY1, SRF AND MSX-1 IN TRANSIENT TRANSFECTION ANALYSIS

Potent viral promoters/enhancers are often used to achieve high level expression of ectopic genes in transient transfection analysis. By usi ng a GAL4-responsive transcription assay system, we show-that the use of potent eukaryotic expression vectors can lead to biased transcripti onal effects. Three functionally diverse transcription factors, YY1, S RF and Msx-1, were examined and each was found to exhibit a strong tra nsrepression function in the context of the DNA binding domain of GAL4 when expressed from the cytomegalovirus (pCMV) or simian virus 40 (pS V) promoters/enhancers. An internal 15 amino acid domain of YY1 mediat ing transrepression in the viral promoter setting was identified. This GAL4-mediated transcriptional repression could, however, be completel y relieved by using the yeast alcohol dehydrogenase promoter (pADH) to drive gene expression, which is similar to 100-fold weaker than canon ical pCMV and pSV in cultured mammalian cells. In addition, low level expression achieved with the pADH vector unveiled the intrinsic transa ctivation functions of YY1 and SRF previously not observed with the GA L4 assay system. Our results highlight a potential pitfall in conventi onal pCMV- and pSV-based transfection assays and suggest that the use of a low level expression system may be preferable in most transcripti onal analysis.