The phytohormone auxin controls numerous plant growth and developmental processes, including many traits of agronomic importance in crops. Thus, engineering endogenous auxin activity has tremendous potential for changing plant architecture by design. Yet this potential remains untapped due to pleiotropic effects associated with altering auxin levels. Auxin signals are locally translated to gene expression changes underlying growth and development by ARF transcription factors, whose DNA-binding mechanisms have recently been uncovered.
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.
Aim of the project
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.
In this project, I will engineer DNA-binding specificity in the Arabidopsis ARF5 protein to rewire endogenous auxin responses. Firstly, by systematically replacing DNA-contacting amino acids, I will generate ARF5 mutant proteins with novel or altered DNA-binding specificity. This resource will help reveal the elusive “code” underlying specific protein-DNA interactions and target gene selection. Secondly, by introducing mutant ARF5s into Arabidopsis, I will determine how intrinsic protein properties translate to DNA-binding properties in plants, and determine the developmental consequences of rewiring the auxin response. Analysing DNA-binding specificity of such mutant ARF5 proteins, as well as phenotypic analysis of the mutant plants, will provide an important proof of concept for inducing plant development adjustments through targeted modification of ARF-DNA interactions, heralding the foundation for plant shape by design.
Do you have any questions about DNA-binding of ARF proteins, or would you like to join us as a student researcher? Please contact us.