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

We have investigated the basis for the striking difference between the broa d DNA sequence selectivity of the c-Myb transcription factor minimal DNA-bi nding domain R2R3 in vitro and the more restricted preference of a R(2)R(3) VP16 protein for Myb-specific recognition elements (MREs) in a Saccharomyce s cerevisiae transactivation system. We show that sequence discrimination i n 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) w ere also included in the study. All of the tested Myb proteins displayed ve ry 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 selectiv ity was highest at low expression levels of Myb effector proteins. However, the discrimination between MRE variants was rapidly lost with high input l evels of effector plasmid. In c-Myb-expressing K-562 cells, the high degree of MRE selectivity was retained, thereby confirming the relevance of the r esults obtained in the yeast system. These data suggest that the MRE select ivity of c-Myb is an intrinsic property of only the R2R3 domain itself and that the transactivation response of a specific MRE in vivo may be highly d ependent on the expression level of the Myb protein in the cell.