The Regulatory Specificity of a Homeodomain Protein is Determined by Unique DNA-Binding Sequences

Homeodomain (HD) proteins comprise a large family of evolutionarily conserved transcription factors (TFs) having diverse developmental functions, yet they paradoxically recognize very similar DNA sequences. To investigate how HDs control cell-specific gene expression patterns, we determined the DNA binding specificities of a broad range of HDs critical for Drosophila embryonic mesoderm development. These studies revealed particular sequences that are bound by one HD and not by others. Such HD-preferred binding sites are overrepresented in the noncoding regions of genes that are regulated by the corresponding HD. Moreover, we show at single-cell resolution in intact embryos that the HD Slouch (Slou) controls myoblast gene expression through unique DNA sequences that are preferentially bound by Slou. These findings demonstrate that the sequence of a HD-binding site dictates which HD family member binds to and regulates a particular enhancer. This represents a novel mechanism for how cell type-specific TFs induce the distinct genetic programs of individual embryonic cells. Mesodermal cells were profiled from wild type, slou gain-of-function and msh gain-of-function backgrounds.