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 using a GAL4-responsive transcription assay system, we show that the use of potent eukaryotic expression vectors can lead to biased transcriptional effects. Three functionally diverse transcription factors, YY1, SRF and Msx-1, were examined and each was found to exhibit a strong transrepression function in the context of the DNA binding domain of GAL4 when expressed from the cytomegalovirus (pCMV) or simian virus 40 (pSV) promoters/enhancers. An internal 15 amino acid domain of YY1 mediating transrepression in the viral promoter setting was identified. This GAL4-mediated transcriptional repression could, however, be completely relieved by using the yeast alcohol dehydrogenase promoter (pADH) to drive gene expression, which is approximately 100-fold weaker than canonical pCMV and pSV in cultured mammalian cells. In addition, low level expression achieved with the pADH vector unveiled the intrinsic transactivation functions of YY1 and SRF previously not observed with the GAL4 assay system. Our results highlight a potential pitfall in conventional pCMV- and pSV-based transfection assays and suggest that the use of a low level expression system may be preferable in most transcriptional analysis.
Lee
T
T
Department of Biochemistry, SUNY at Buffalo, 140 Farber Hall, 3435 Main Street, Buffalo, NY 14214-3000, USA. chunglee@acsu.buffalo.edu
Bradley
M E
ME
Walowitz
J L
JL
D000426:Alcohol Dehydrogenase / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism*; D050981:Erythroid-Specific DNA-Binding Factors; D005822:Genetic Vectors; D018398:Homeodomain Proteins / Q000378:metabolism*; D051957:MSX1 Transcription Factor; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D029701:Saccharomyces cerevisiae Proteins; D026362:Serum Response Factor; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection; D050986:YY1 Transcription Factor
D017931:DNA Primers; D004268:DNA-Binding Proteins; D050981:Erythroid-Specific DNA-Binding Factors; C052130:GAL4 protein, S cerevisiae; D018398:Homeodomain Proteins; D051957:MSX1 Transcription Factor; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; D026362:Serum Response Factor; D014157:Transcription Factors; D050986:YY1 Transcription Factor; C494699:Yy1 protein, mouse; D000426:Alcohol Dehydrogenase