Brachyury proteins regulate target genes through modular binding sites in a cooperative fashion

Brachyury proteins, a conserved subgroup of the T domain transcription factors, specify gut and posterior mesoderm derivatives throughout the animal kingdom. The T domain confers DNA-binding properties to Brachyury proteins, but little is known how these proteins regulate their target genes. We characterized a direct target gene of the Drosophila Brachyury-homolog Brachyenteron. Brachyenteron activates the homeobox gene orthopedia in a dose-dependent manner via multiple binding sites with the consensus (A/G)(A/T)(A/T)NTN(A/G)CAC(C/T)T. The sites and their A/T-rich flanking regions are conserved between D. melanogaster and Drosophila virilis. Reporter assays and site-directed mutagenesis demonstrate that Brachyenteron binding sites confer in part additive, in part synergistic effects on otp transcription levels. This suggests an interaction of Brachyenteron proteins on the DNA, which we could map to a conserved motif within the T domain. Mouse Brachyury also interacts with Brachyenteron through this motif. We further show that the Xenopus and mouse Brachyury homologs activate orthopedia expression when expressed in Drosophila embryonic cells. We propose that the mechanisms to achieve target gene expression through variable binding sites and through defined protein-protein interactions might be conserved for Brachyury relatives.