EAGER: Decoding Maize Transcription Factor-Mediator Interactions: A Breakthrough Tool for Precision Gene Regulation

The translation of genotype into phenotype relies on tightly regulated gene expression, orchestrated by transcription factors (TFs) tethered to cis-regulatory elements and interacting with the multi-subunit Mediator (MED) complex, which plays a crucial role in conveying regulatory information from TFs to the transcriptional machinery. TFs have been widely utilized to manipulate plant metabolism, stress responses, and developmental processes; however, unintended regulatory activities have often led to unpredictable and undesired outcomes. Thus, achieving precise control over the regulatory activity of TFs would represent a major breakthrough in leveraging TFs for the targeted manipulation of genetic programs. The goal of this EAGER project is to leverage existing tools and functional genomics resources to design synthetic TFs that bind uniquely to engineered MED partners in maize, effectively rewiring the transcriptional machinery, creating a dynamic and responsive system for finely tuned gene regulation. If successful, this project would provide a novel alternative to existing strategies for reprogramming transcriptional specificity, which could be broadly applicable for use in synthetic biology projects in plants as well as in animal systems. Precise control of gene expression remains a central challenge in plant synthetic biology. The objective of this potentially transformative, yet high-risk proof of concept project is to develop Mediator–Engineered Regulator Interaction Tuning (MERIT), a novel synthetic biology platform that enables precise, programmable control of gene expression by engineering specific interactions between maize TFs with altered specificity and the MED complex, a central integrator of transcription. Specific objectives are to: 1) elucidate the maize TF-MED interaction space using a high-throughput yeast two-hybrid (Y2H) interaction screen between cloned MED subunits and maize TFs identified and characterized with previous NSF funding; 2) strategically introduce targeted mutations into select TFs (referred as TF*), designed to disrupt their interaction with MED; 3) identify complementary mutations in the interacting MED subunit (MED*) that restore interaction specifically with TF* while avoiding interactions with wild-type TF; and, 4) introduce selected compensatory TF*-MED* pairs into maize protoplasts to assess their transcriptional activity, tested alongside the wild-type counterparts to evaluate functional specificity. In addition to the broader impacts of the tools generated for the plant genomics research community and for crop improvement through biotechnology, this project will continue to provide research training and career mentoring for undergraduate and graduate students. All project outcomes will be available through long-term repositories and peer-reviewed manuscripts. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.