A valid strategy for precise identifications of transcription factor binding sites in combinatorial regulation using bioinformatic and experimental approaches

Abstract

BackgroundTranscription factor (TF) binding sites (cis element) play a central role in gene regulation, and eukaryotic organisms frequently adapt a combinatorial regulation to render sophisticated local gene expression patterns. Knowing the precise cis element on a distal promoter is a prerequisite for studying a typical transcription process; however, identifications of cis elements have lagged behind those of their associated trans acting TFs due to technical difficulties. Consequently, gene regulations via combinatorial TFs, as widely observed across biological processes, have remained vague in many cases.ResultsWe present here a valid strategy for identifying cis elements in combinatorial TF regulations. It consists of bioinformatic searches of available databases to generate candidate cis elements and tests of the candidates using improved experimental assays. Taking the MYB and the bHLH that collaboratively regulate the anthocyanin pathway genes as examples, we demonstrate how candidate cis motifs for the TFs are found on multi-specific promoters of chalcone synthase (CHS) genes, and how to experimentally test the candidate sites by designing DNA fragments hosting the candidate motifs based on a known promoter (us1 allele of Ipomoea purpurea CHS-D in our case) and applying site-mutagenesis at the motifs. It was shown that TF-DNA interactions could be unambiguously analyzed by assays of electrophoretic mobility shift (EMSA) and dual-luciferase transient expressions, and the resulting evidence precisely delineated a cis element. The cis element for R2R3 MYBs including Ipomoea MYB1 and Magnolia MYB1, for instance, was found to be ANCNACC, and that for bHLHs (exemplified by Ipomoea bHLH2 and petunia AN1) was CACNNG. A re-analysis was conducted on previously reported promoter segments recognized by maize C1 and apple MYB10, which indicated that cis elements similar to ANCNACC were indeed present on these segments, and tested positive for their bindings to Ipomoea MYB1.ConclusionIdentification of cis elements in combinatorial regulation is now feasible with the strategy outlined. The working pipeline integrates the existing databases with experimental techniques, providing an open framework for precisely identifying cis elements. This strategy is widely applicable to various biological systems, and may enhance future analyses on gene regulation.

Phylogeny and Expression Analyses Reveal Important Roles for Plant PKS III Family during the Conquest of Land by Plants and Angiosperm Diversification

“…Except for the missing values, genes expressed in at least one tissue all had a complete motif that was suitably located ( Figures 5B,C ). As expected, the most conserved motif of clade A genes embraced adjacent MYB and bHLH binding sites, two transcription factors of the MBW transcriptional regulatory complex, which have been experimentally verified in several species (Wang et al, 2013; Zhu et al, 2015). We then analyzed the conserved motif in each sub-clade.…”

MYB118 Represses Endosperm Maturation in Seeds of Arabidopsis

“…MYB118 encodes an R2R3-type MYB TF harboring two adjacent R repeats, which is a common feature of plant MYBs. Although many R2R3-MYB TFs recognize AC elements (DNA motifs enriched in adenosine and cytosine residues), the range of R2R3-MYB binding sites remains poorly defined in planta (Prouse and Campbell, 2012;Wang et al, 2013). Our observations suggest that the TAACGG element is part of the in vivo MYB118 binding site.…”

Deciphering the Role of various cis-acting regulatory elements in controlling SamDC gene expression in Rice

“…In plants the MYB transcription factors contains R2 and R3 domains and the binding sequence varies from four to 14 nucleotides. 12 In Arabidopsis it has been shown that the β-glucoronidase gene driven by AtMYB2 promoter was inducible by osmotic stresses. 13 The upstream analysis of SamDC promoter showed the presence of W-Box elements and MYB regulatory cis elements (TAACTG).…”