Identification of nucleotide preferences in DNA sequences recognised specifically by c-Ets-1 protein

The protooncogene Ets-1 is a member of the c-Ets family of genes originally identified through their sequence homology to the v-ets gene of the avian erythroblastosis virus E26. Ets-like factors are characterised by a conserved 85 amino acid domain which appears to be essential for binding to purine rich DNA sequences. Sequences binding to Ets-1 were selected from a random oligonucleotide pool by immunoprecipitation and amplified using the Polymerase Chain Reaction. Oligonucleotides enriched by this procedure were cloned in plasmids and sequenced. Alignment of DNA sequences revealed GG-AA and GGAT cores at about a 1.4:1 ratio. Preferred sequences were identified both 5' and 3' of the GGAW core, extending the binding site to ACMGGAWRTT. Analysis of the flanking sequences associated with GG-AA and GGAT cores revealed differences which may have compensated for the generally lower affinity of binding sites containing a GGAT core. Lastly mutational analysis of one particular Ets-1 binding site was used to establish the relative importance for binding of some nucleotides within the core and to show that Ets-1 and the closely related Ets-2 proteins bind to similar sequences.