Abstract
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.
Highlights
The control of gene expression is crucial for proper development in any living organism
Transcriptional control is mediated by short DNA sequences located in gene promoters that are bound by transcription factors (TFs)
LIP1 orthologous gene promoters from several plant species belonging to the Brassicaceae family (A. thaliana, Sisymbrium irio, Sinapis arvensis, Capsella rubella, Descuriainia sophia, Brassica oleracea) were amplified by using a PCRbased approach with degenerated oligonucleotides
Summary
The control of gene expression is crucial for proper development in any living organism. Combinatorial control driven by different cis-elements and their corresponding TF proteins at a given promoter is an important but not well understood area of plant gene regulation [3]. Non-coding sequences of orthologous genes diverge rapidly during evolution, except for those that are functionally important. This divergence in promoter sequences can be exploited to identify conserved sequences important for the regulation of gene expression, which reduces the need for time-consuming promoter analyses involving random deletions to generate promoter variants. The sequences identified by phylogenetic shadowing contain a combination of several cis-elements, providing valuable information on the conservation of the core and flanking sequences and pinpointing the different elements to consider when studying the regulation of a given promoter
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