Sequence selectivity of c-Myb in vivo. Resolution of a DNA target specificity paradox

• Published in: The Journal of biological chemistry 1999-07, Vol.274 (31), p.21986-21994
• Main Authors: Andersson, K B, Berge, T, Matre, V, Gabrielsen, O S
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; Binding Sites; Cell Line; COS Cells; DNA-Binding Proteins - metabolism; Genes, Reporter; Humans; K562 Cells; Luciferases - genetics; Molecular Sequence Data; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - metabolism; Promoter Regions, Genetic; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - metabolism; Proto-Oncogene Proteins c-myb; Recombinant Fusion Proteins - biosynthesis; Saccharomyces cerevisiae - genetics; Substrate Specificity; Trans-Activators - genetics; Trans-Activators - metabolism; Transcriptional Activation; Transfection
• ISSN: 0021-9258
• DOI: 10.1074/jbc.274.31.21986

We have investigated the basis for the striking difference between the broad DNA sequence selectivity of the c-Myb transcription factor minimal DNA-binding domain R(2)R(3) in vitro and the more restricted preference of a R(2)R(3)VP16 protein for Myb-specific recognition elements (MREs) in a Saccharomyces cerevisiae transactivation system. We show that sequence discrimination in yeast is highly dependent on the expression level of Myb effector protein. Full-length c-Myb and a C-terminally truncated protein (residues 1-360) were also included in the study. All of the tested Myb proteins displayed very similar DNA binding properties in electrophoretic mobility shift assays. Only minor differences between full-length c-Myb and truncated c-Myb(1-360) were observed. In transactivation studies in CV-1 cells, the MRE selectivity was highest at low expression levels of Myb effector proteins. However, the discrimination between MRE variants was rapidly lost with high input levels of effector plasmid. In c-Myb-expressing K-562 cells, the high degree of MRE selectivity was retained, thereby confirming the relevance of the results obtained in the yeast system. These data suggest that the MRE selectivity of c-Myb is an intrinsic property of only the R(2)R(3) domain itself and that the transactivation response of a specific MRE in vivo may be highly dependent on the expression level of the Myb protein in the cell.