Hox Control of Morphogenesis via Co-Evolution of Numbers and Affinities of Hox Binding Sites with Transcription Factor Concentrations

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The Hox family of transcription factors (TFs) drive morphological diversification of body segments along the anterior-posterior (A/P) axis through a high level of transcriptional specificity. The precise assignment of morphological features along the A/P axis in vivo contrasts with the poor DNA binding stringency of Hox TFs in vitro, as they recognize frequently encountered DNA sequences. It is evident that other regulatory proteins and differences in binding sequences, in particular low-affinity sites, play a critical role in conferring specificity to Hox proteins. Despite increasing our understanding of Hox in vivo function, the emergence of Hox low-affinity binding sites also opened new questions. Specifically, the contribution of high- and low-affinity binging sites to Hox target gene regulation and morphogenesis is unclear and whether other homeodomain TFs use the same mechanism of increasing specificity by decreasing affinity and thereby contribute to the evolution of different types of Hox binding sites.To answer these questions we have established a model, the regulation of the AP-2 gene by the Hox TF Deformed (Dfd). This regulatory interaction depends on a combination of low- and high-affinity Dfd binding sites, controls a morphological feature, the maxillary cirri, and occurs in cells expressing other homeodomain TFs. We will dissect how the Hox TF Dfd reads the regulatory information encoded in the AP-2 enhancer by interfering with the number and affinity of Hox sites and modulate the regional expression of Dfd in reporter-based assays. We will test the contribution of homeodomain TFs interacting with similar consensus sequences as Dfd to AP-2 transcription using domain-specific over-expression and knock-downs. We will analyse the contribution of these features to morphogenesis by testing the effect of their modifications on maxillary cirri formation in a UAS-GAL4 rescue setting and by engineering the endogenous locus using the CRISPR/Cas9. Finally, we will dissect the regulatory input assisting Dfd in controlling AP-2 expression by identifying all proteins interacting with the AP-2 CRM using a enhancer-specific proteomics approach, termed EnhanceProteome. Analysis of individual Dfd co-regulators in future will allow us to elucidate how they contribute in combination with Dfd to AP-2 expression in the maxillary segment.This study will provide critical insights into how Hox TFs control their target genes and thus morphogenesis, as it takes low- and high-affinity binding sites as well as their clustering into consideration and will test their importance for organismal fitness. This study will also make fundamental contributions to the affinity-specificity trade-off model and extend it to the whole class of homeodomain TFs. Finally, it will establish a versatile tool to isolate the full complement of proteins interacting with a specific enhancer fragment, which will open new avenues in the field of transcriptional regulation.