Identification of Arabidopsis Transcriptional Regulators by Yeast One-Hybrid Screens Using a Transcription Factor ORFeome.

Genetic and molecular approaches revealed that the circadian clock network structure is comprised of several interlocked positive and negative transcriptional feedback loops. The network evolved to sense and integrate inputs from environmental cues to adjust daily rhythms in physiological processes. Compiling evidence indicates that part of this regulation happens at the transcriptional level through subtle adjustments in the expression of core clock genes. Thus, to better understand the network and identify the molecular mechanisms of clock input pathways, it is imperative to determine how core clock genes are regulated. For this purpose we developed reagents for an unbiased approach to identify transcription factors (TFs) interacting with the promoters of core clock genes. At the center of this approach lies the yeast one-hybrid (Y1H) assay in which a pool of proteins fused to the GAL4 transcriptional activation domain are tested for their ability to interact with a selected promoter fragment in yeast cells. Taking advantage of the fact that Arabidopsis TF genes are well annotated, we generated a comprehensive TF clone collection (TF ORFeome) and used it to replace the standard cDNA pool strategy traditionally used in Y1H screens. The use of this TF clone collection substantially accelerates the comprehensive discovery of promoter-specific DNA binding activities among all Arabidopsis TFs. Considering that this strategy can be extended to the study of the promoter interactome of any Arabidopsis gene, we developed a low throughput protocol that can be universally implemented to screen the ~2000 TF clone library.