Speeding cis-trans regulation discovery by phylogenomic analyses coupled with screenings of an arrayed library of Arabidopsis transcription factors

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.

Figure 1. Identification of functionally relevant promoter cis-elements in a GDSL-lipase gene from A. thaliana.

(A) The promoter of the A. thaliana GDSL-lipase gene (At5g45670) and their orthologous promoters in other Brassicaceae species were subjected to in silico analysis to identify conserved sequences (shaded boxes). Although over 1 Kb of each promoter was analyzed, significantly conserved sequences were found only along the first 500 bp upstream of the translation start site (TSS). The sequence with the highest degree of conservation (83% identity) is represented as a dark blue box and spans 50 bp (LIP1 element). (B) A binary plasmid (pYRO) containing a minimal promoter fused to the luciferase reporter gene (control) was used to clone four copies of the 50 bp sequence (4xLIP1) and both constructs were used to produce A. thaliana transgenic plants. Luciferase activity was quantified in vivo from transgenic seeds 24 h after imbibition. Average values and standard errors from 10 independent lines for each construct (20 seeds/line) are shown.

Figure 2. Flowchart for the yeast screening procedure for the arrayed TF library in 96-well format.

TF library and bait clones are grown on plates with their corresponding auxotrophic media. These plates were used to inoculate either 96-well plates (TF library; preys) or Erlenmeyer flasks (cis-element in pTUY1H; bait) containing YPAD and incubated overnight. Bait and preys were then mixed and incubated for 48 h without shaking to allow mating. Mated cells were used to inoculate another set of 96-well plates containing diploid selection media (DOB-L-W). After incubation for 24 h, diploid cells were replicated onto diploid and screening (DOB-L-W-H ±3-AT) plates. Positives were visible after 2 to 5 days of growth. Hours of labour per person are indicated for each step of the protocol.

Figure 3. Yeast one hybrid screening with the LIP1-pTUY1H construct.

Growth of diploid cells at different concentrations of 3-AT from clones showing positive (3-H3) and negative (15-G9) interactions in the screening. Three serial dilutions of diploid cells from saturated cultures were plated.

Figure 4. Specificity and in planta relevance of the AtML1-LIP1 element interaction and screening with the IPS1-element.

(A) Alignment of the LIP1 element (LIP1wt) from different Brassicaceae species. A conserved L1-box sequence putatively bound by AtML1 and other HD-ZIP TFs is shaded. A mutated version of the Arabidopsis LIP1 element with 2 bp changes in the L1-box core sequence was cloned into the pTUY1H plasmid (LIP1-L1mut). (B) AtML1 specifically binds to the L1-box sequence of the LIP1 element. Yeast strains containing either the LIP1 element (WT) or a 2 bp mutation in the L1-box (mut), were mated to strains containing the AD-AtML1 or AD-GFP (negative control) constructs. Diploid cells were grown on auxotrophic media with increasing concentrations of 3-AT. (C) The LIP1 element is an AtML1 target in planta. Leaves from transgenic plants carrying the 4xLIP1-58F8-pYRO construct were bombarded with an empty plasmid (top images) as a control or with a 35S:AtML1 construct (bottom images). Bioluminescence images from three independent experiments are shown. (D) A promoter fragment from the IPS1 gene containing a phosphate starvation responsive element is bound by PHR1 (R1MYB) but not by other R1MYB TF. A strain containing the IPS1 promoter fragment was mated to strains containing the PHR1 or another family protein (negative control). Diploid cells were grown as in part (B).

Figure 5. HD-ZIP subfamily IV and MYB-CC proteins show differential binding capabilities.

(A) Phylogenetic tree of HD-ZIP class IV TF proteins from A. thaliana constructed using the Phylogeny.fr platform. TFs used in part (B) are indicated by green and red colored circles. (B) Yeast strains containing either the LIP1 element (WT) or a 2 bp mutation in the L1-box (mut), were mated to strains containing the AD-AtML1, AD-GL2 or AD-HDG10 constructs. Diploid cells were grown on screening plates with increasing concentrations of 3-AT. Only the AtML1 protein is able to activate the reporter gene by binding to the WT element. (C) Phylogenetic tree of MYB-CC TF proteins from A. thaliana modified from a tree published elsewhere . A dotted line separates Group 1 from Group 2 subfamily members characterized by having the MYB-CC domain at C or N-terminal position, respectively. TFs used in part (D) are indicated by green and red colored circles. (D) A yeast strain containing the IPS1 element was mated to strains containing the AD-PHR1 or AD-MYB-CC TFs. Diploid cells were grown on diploid and screening plates. Only PHR1 and PHR1-like proteins belonging to Group 1 were able to activate the reporter gene .Three serial dilutions of diploid cells were spotted.