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Transcriptional regulation is an important mechanism underlying gene expression and has played a crucial role in evolution. The number, position and interactions between  cis -elements and transcription factors (TFs) determine the expression pattern of a gene. To identify functionally relevant  cis -elements in gene promoters, a phylogenetic shadowing approach with a lipase gene ( LIP1 ) was used. As a proof of concept, in silico analyses of several  Brassicaceae LIP1  promoters identified a highly conserved sequence ( LIP1  element) that is sufficient to drive strong expression of a reporter gene in planta. A collection of ca. 1,200  Arabidopsis thaliana  TF open reading frames (ORFs) was arrayed in a 96-well format (RR library) and a convenient mating based yeast one hybrid (Y1H) screening procedure was established. We constructed an episomal plasmid (pTUY1H) to clone the  LIP1  element and used it as bait for Y1H screenings. A novel interaction with an HD-ZIP (AtML1) TF was identified and abolished by a 2 bp mutation in the  LIP1  element. A role of this interaction in transcriptional regulation was confirmed in planta. In addition, we validated our strategy by reproducing the previously reported interaction between a MYB-CC (PHR1) TF, a central regulator of phosphate starvation responses, with a conserved promoter fragment ( IPS1  element) containing its cognate binding sequence. Finally, we established that the  LIP1  and  IPS1  elements were differentially bound by HD-ZIP and MYB-CC family members in agreement with their genetic redundancy in planta. In conclusion, combining in silico analyses of orthologous gene promoters with Y1H screening of the RR library represents a powerful approach to decipher  cis-  and  trans -regulatory codes.

Speeding Cis-Trans Regulation Discovery by Phylogenomic Analyses Coupled with Screenings of an Arrayed Library of Arabidopsis Transcription Factors