DIMR, a Yeast-Based Synthetic Reporter System for Probing Oligomeric Transcription Factor DNA Binding

Abstract

Transcription factors (TFs) are fundamental components of biological regulation, facilitating the basal and differential gene expression necessary for life. TFs exert transcriptional regulation through interactions with both DNA and other TFs, ultimately influencing the action of RNA polymerase at a genomic locus. Current approaches are proficient at identification of binding site requirements for individual TFs, but few methods have been adapted to study oligomeric TF complexes. Further, many approaches that have been turned toward understanding DNA binding of TF complexes, such as electrophoretic mobility shift assays, require protein purification steps that can be burdensome or scope-limiting when considering more exhaustive experimental design. In order to address these shortfalls and to facilitate a more streamlined approach to understanding DNA binding by TF complexes, we developed the DIMR (Dynamic, Interdependent TF binding Molecular Reporter) system, a modular, yeast-based synthetic transcriptional activity reporter. As a proof of concept, we focused on the NUCLEAR FACTOR-Y (NF-Y) family of obligate heterotrimeric TFs in Arabidopsis thaliana. The DIMR system was able to reproduce the strict DNA-binding requirements of an experimentally validated NF-YA2/B2/C3 complex with high fidelity, including recapitulation of previously characterized mutations in subunits that either break NF-Y complex interactions or are directly involved in DNA binding. The DIMR system is a novel, powerful, and easy-to-use approach to address questions regarding the binding of oligomeric TFs to DNA.\n\nOne sentence summaryThe DIMR system provides an accessible and easy-to-use platform to elucidate DNA binding and transcriptional regulatory capacity of oligomeric transcription factor complexes