Robust Hox-Mediated Transcriptional Regulation Utilizes a Combination of Flexible Binding Site Composition and Rigid Grammar. Juli Uhl, Lisa Gutzwiller, Arif Ghasletwala, Brian Gebelein. Developmental Biology, Cincinnati Children's Hospital, Cincinnati, OH.

   Designer enhancers that selectively direct gene expression would be a useful research tool, but to create these we must first define the rules of enhancer organization. Many transcription factors bind common DNA motifs yet regulate gene expression in a tissue-specific manner. In Drosophila, the Abdominal-A Hox factor (AbdA) directs formation of specialized metabolic cells by activating rhomboid in a subset of sensory organ precursors, while in nearby cells AbdA restricts leg primordia to the thorax by repressing Distal-less. The enhancers for rhomboid (RhoA) and Distal-less (DMXR) both contain binding sites for AbdA, Extradenticle (Exd), and Homothorax (Hth), yet the order and spacing of the sites differs. In addition, RhoA requires a nearby dPax2 site while DMXR requires a nearby Sloppy-Paired (Slp) site for proper transcriptional outcomes. Here, we compare RhoA and DMXR using quantifiable reporter and DNA binding assays to understand the grammar of Hox-regulatory enhancers. Notably, we found that the Hox, Exd, and Hth motif in RhoA is capable of repression in place of that in DMXR. Moreover, we discovered that neither RhoA nor DMXR require Hth binding for in vivo activity despite the crucial role Hth plays in cooperative complex formation in vitro. Finally, we found that RhoA remains active when the dPax2 site is moved, but DMXR repression requires a particular orientation and spacing of Slp motifs relative to the nearby Hox motif. While the relationship between Hox factors and cofactors is complex, these data suggest that; 1-Functional gene regulation does not require cooperative binding of Hox and Exd/Hth. 2-There are two types of Hox:cofactor interactions; flexible dPax2:Hox configuration and inflexible Slp:Hox configuration. 3- Hox/Exd/Hth motifs are context-sensitive activators or repressors. Together, these data suggest that a combination of rigid grammar and flexible configuration of motifs regulates robust Hox-mediated gene expression.