Deciphering the rules of genome binding specificity: de-novo design of intrinsically disordered regions directing transcription factor binding.

• Applicant: Professor Dr. Felix Jonas
• Subject Area: General Genetics and Functional Genome Biology
• Term: since 2024
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 550609939

Transcription factor (TF) proteins orchestrate gene expression within cells by binding to specific gene regulatory regions. To function reliably, TFs need to locate their target sites within large genomes specifically and rapidly. Neither the specificity, nor the speed of the TF-target search process are readily explained within existing models. The proposed project follows our recent results, pointing at intrinsically disordered regions (IDRs) of TFs as the key for understanding both the specificity and the speed of this search processes. Previously, we showed that IDRs can direct TF binding in genomes and provided some initial indications of the underlying molecular mechanisms and the associated sequence grammars. Most notably, the role of IDRs in directing DNA binding preferences depends on multiple determinants and these are encoded by the IDR’s bulk features such as amino acid composition rather than sequence motifs. Still, the basis of this interaction remained unclear. In this project, we will take a synthetic biology approach to define the key IDR sequence features directing binding preferences. First, we will use bioinformatics to define recurrent (and conserved) features of TF IDRs. These features will then be used for the de-novo design of IDR archetypes: IDRs that implement these design features with minimal sequence complexity. We will verify and analyze the activity of these archetypes using genome-wide profiling. Of note, in preliminary work, we successfully designed two such IDR archetypes that displays specific genomic preferences, confirming the feasibility of our approach. Once obtained, we will proceed to expand this set of genome-binding IDRs through quantitative changes in their design features. This will define the quantitative relation between IDR bulk features and the choice of IDR-bound target sites, enabling predictive IDR designs of bespoke specificity. Finally, in our third aim, we will combine the designed IDRs with DNA binding domains (DBDs) and study their interaction. This will advance our understanding of the IDR-DBD interaction in native TFs and further generate IDR-based synthetic TFs of pre-specified binding preferences. In conclusion, our project embraces de-novo designed IDRs for testing a fundamental unknown in the field of gene regulation: how TFs locate their specific binding sites in large genomes. Thereby, we overcome the complexities associated with natural sequences that evolved to full-fill multiple functions. We can thus focus on the design features critical for binding specificity. Through our three aims, we will define the IDR bulk features that enable distinctive binding preferences, reveal how tuning and combining these features can adjust specificity, and describe the rules governing IDR-DBD interactions. Together, this project will form the basis for future mechanistic studies of TF IDRs, and the generation of new synthetic TF for biotechnological applications.

• DFG Programme: Research Grants
• International Connection: Israel
• International Co-Applicant: Professorin Dr. Naama Barkai