Tackling the Hox Specificity Problem: Decoding DNA Binding and Protein Interaction Properties of CRISPR Engineered Hox Chimeras

• Applicant: Professorin Dr. Ingrid Lohmann
• Subject Area: Developmental Biology ; General Genetics and Functional Genome Biology
• Term: since 2010
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 158148543

Hox proteins, known for their highly conserved DNA-binding homeodomain, exert a significant influence on developmental trajectories by intricately defining segmental identities along the anterior-posterior axis. Despite their nuanced in vivo functions, Hox transcription factors (TFs) show a propensity for similar binding sequences in vitro, leading to the enigmatic Hox paradox. While certain aspects of this phenomenon have been elucidated, numerous questions remain, including the divergence of transcriptional profiles between closely related Hox proteins and the determinants that govern this specificity. We have previously shown that the anterior Hox protein Deformed (Dfd) activates its target gene AP-2 via a Dfd-specific enhancer, which distinguishes it from the closely related Hox protein Sex combs reduced (Scr). Using Dfd and Scr as models, we now seek to answer these questions by investigating the DNA binding affinity, transcriptional responses (both activation and repression) and protein-protein interaction dynamics of Dfd and Scr in their natural physiological context. In particular, we will compare these results with chimeric Dfd and Scr proteins to clarify the role of the homeodomain in mediating specificity in the in vivo context. To this end, we will use CRISPR-mediated genome editing to replace the entire homeodomain of Dfd or Scr with corresponding segments of the neighbouring Hox gene to generate Dfd-ScrHD and Scr-DfdHD chimeras. This approach ensures precise expression of Dfd and Scr protein variants in embryos that reflect the spatio-temporal and quantitative aspects of wild-type Dfd and Scr. This is particularly important as previous results with misexpression of Hox proteins and chimeric Hox proteins lead to abnormally high TF expression levels. In addition, we will identify new interaction partners of Dfd and Scr using the UltraID (proximity-dependent labelling) method in the native environment to uncover the intricate mechanisms that determine their functional specificity. With the results of this research, we aim to modify the AP2-377 enhancer, which has been shown to be selective for Dfd, to confer specificity to Scr by modifying Hox as well as cofactor DNA binding motifs in an manner informed by this research. Overall, this study will provide unprecedented insight into how closely related Hox proteins precisely regulate target genes in the organism's cellular milieu. Importantly, this study may also shed light on the evolutionary implications of genetic re-organisation and domain exchange within TFs and thus has the potential to revolutionize our understanding of gene regulation and developmental biology, with far-reaching implications for both basic and applied science.

• DFG Programme: Research Grants