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 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.
Department of Biochemistry, University of Oslo, N-0316 Oslo 3, Norway. k.b.andersson@ioks.uio.no
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D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D004268:DNA-Binding Proteins / Q000378:metabolism; D017930:Genes, Reporter; D006801:Humans; D020014:K562 Cells; D008156:Luciferases / Q000235:genetics; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism*; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D012441:Saccharomyces cerevisiae / Q000235:genetics; D013379:Substrate Specificity; D015534:Trans-Activators / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D011518:Proto-Oncogene Proteins; D020598:Proto-Oncogene Proteins c-myb; D011993:Recombinant Fusion Proteins; D015534:Trans-Activators; D008156:Luciferases