One of the central questions in molecular biology is how transcription factors (TFs) bind DNA, because only by understanding this question can we predict how we can use these pathways to make our crop plants stronger and healthier. Although we have seen great strides towards an answer, there are still large gaps in our understanding. For instance, with modern tools we have been able to unravel the specific DNA-binding motifs for many TF families. The fruits of this labor can be found in the JASPAR database, a rich source of DNA-binding data. But even by knowing thousands upon thousands of DNA-binding motifs for this wide variety of transcription factors, we are still not close to solving a simple question: how? How are these transcription factors binding these specific motifs, and are there a set of central rules that determine their specificity?
One way to determine how specific transcription factors can interact with their specific DNA-motif is by solving their crystal structure. Each time we solve the exact atomic forces that enable a specific TF-DNA binding interaction we increase our understanding. But with a catch, because each TF is different from the others, and will bind in a completely unique way. We can even see differences between members of the same TF family. So while we gain valuable knowledge from these studies, they are still limited in answering whether or not there are central rules determining their specificity.
That is the focus of my project: investigating the central tenets of DNA-binding, and specifically the Auxin Response Factors (ARFs). Crystal structures of the ARFs have revealed the amino acids that interact with the DNA, and these happen to occur in flexible loops of the protein. That is where most projects would stop. But I will use this flexibility to change these residues at will to decode the ARF DNA-binding. By systematically mutating each amino acid we will gain a rich overview of the effects of certain amino acids on DNA-binding. This knowledge would unlock the potential to hack the ARFs to cause subtle changes in growth and development. Furthermore, this knowledge base and the pipelines created in this project will be invaluable for validating the code of DNA-binding of other TFs. Finally, the research will be a milestone in elucidating the central rules of TF-DNA binding.
Several student projects are available in which you can contribute to this research. If you are interested in bioinformatics work we can model the effects of certain mutations in the crystal structures. If biochemistry is more your thing we can use DNA Affinity Purification sequencing (DAP-seq) for high-throughput screening of ARF-mutations, or we can use bacterial or yeast one hybrids (B1H or Y1H) for deep mutational scanning (DMS). For molecular experience we can transform Marchantia polymorpha with mutated ARFs, and for physiological experience we will phenotype the plants. If you would be interested in any of these projects please contact me and/or prof. Dolf Weijers for current specific projects.