DNA-binding specificities of plant transcription factors and their potential to define target genes

Abstract

Transcription factors (TFs) regulate gene expression through binding to cis-regulatory specific sequences in the promoters of their target genes. In contrast to the genetic code, the transcriptional regulatory code is far from being deciphered and is determined by sequence specificity of TFs, combinatorial cooperation between TFs and chromatin competence. Here we addressed one of these determinants by characterizing the target sequence specificity of 63 plant TFs representing 25 families, using protein-binding microarrays. Remarkably, almost half of these TFs recognized secondary motifs, which in some cases were completely unrelated to the primary element. Analyses of coregulated genes and transcriptomic data from TFs mutants showed the functional significance of over 80% of all identified sequences and of at least one target sequence per TF. Moreover, combining the target sequence information with coexpression analysis we could predict the function of a TF as activator or repressor through a particular DNA sequence. Our data support the correlation between cis-regulatory elements and the sequence determined in vitro using the protein-binding microarray and provides a framework to explore regulatory networks in plants.

Keywords: Arabidopsis; cis-element; coregulation; regulatory network; transcriptional regulation.

Fig. 1.

DNA-binding specificity of plant TFs. Position weight matrix (PWM) representation of top-scoring 8-mers for the TFs indicated. Secondary and tertiary motifs were considered when they differed substantially from primary ones among the lists of top-scoring motifs or after reranking the motifs (Methods). TFs are grouped into families or subfamilies according to a previous classification (1).

Fig. 2.

Binding motifs are associated with DNase I hypersensitive regions. (A) Density plots of TF-binding sites in DNase I hypersensitive sites (DHS) from leaf (Left) and flower (Right) described in ref. . Density of TF-binding sites (red diamonds and line) is measured as the number of sites at each position per number of fragments with that length, along DHS fragments (1 kb showed, centered at the middle nucleotide of each DHS). In blue is represented the average density for 100 randomized PWMs. (B) Density plots of binding sites recognized by HD-ZIP proteins ICU4 (Upper) and ATHB51 (Lower).

Fig. 3.

Evaluation of biological relevance of DNA-motifs from coregulation information. Lists of coregulated genes with the TF-coding genes were scanned for the presence of DNA-motifs obtained in vitro at their promoter regions (1 kb). Frequencies (in %) of genes positively (blue bars) and negatively (red bars) coregulated genes containing the indicated DNA elements are shown. Proportions of genes represented in the ATH1 microarray containing the corresponding elements, and thus representing a random distribution, are represented as green bars. Asterisks indicate the degree of statistical significance in the differences of the proportions indicated as follows: *P < 0.005; **P < 0.05.

Fig. 4.

Mapping of DNA-motifs along upstream and downstream regions. Frequency plots of the average distribution of binding sites at upstream and downstream regions (1 kb) of the genes represented in the ATH1 microarray (green lines), positively coregulated (blues lines), and negatively coregulated (red lines). “0” represents the transcriptional start site in upstream plots, and to stop codon in downstream ones. Plots on the top correspond to binding sites for activators and bottom plots to transcriptional repressors.