Autoregulatory binding sites in the zebrafish six3a promoter region define a new recognition sequence for Six3 proteins

The homeodomain (HD) transcription factor Six3, which is a member of the Six/Sine oculis family, is essential for development of the eyes and forebrain in vertebrates. It has recently been claimed that the HDs of Six3 and other members of the Six family have a common recognition sequence, TGATAC. However, a different recognition sequence including the typical TAAT core motif, which has not yet been fully defined, has also been proposed for the Six3 HD in mice. Our study of the zebrafish orthologue six3a, which has an identical HD, shows that it binds in vitro to multiple TAAT-containing sites within its promoter region. Comparison of the different binding affinities for these sequences identifies three high-affinity sites with a common TAATGTC motif. Notably, this new recognition sequence, which is supported by our analysis of the influence of single-nucleotide substitutions on the DNA-binding affinity, is distinct from all of the DNA-binding specificities previously described in surveys of HDs. In addition, our comparison of Six3a HD binding to the novel TAATGTC motif and the common recognition sequence of Six family HDs (TGATAC) shows very similar affinities, suggesting two distinct DNA-binding modes. Transient reporter assays of the six3a promoter in zebrafish embryos also indicate that the three high-affinity sites are involved in autoregulation. In support of this, chromatin immunoprecipitation experiments show enrichment of Six3a binding to a six3a promoter fragment containing two clustered high-affinity sites. These findings provide strong evidence that the TAATGTC motif is an important target sequence for vertebrate Six3 proteins in vivo.