Computational analysis of auxin responsive elements in the Arabidopsis thaliana L. genome

Abstract

Auxin responsive elements (AuxRE) were found in upstream regions of target genes for ARFs (Auxin response factors). While Chip-seq data for most of ARFs are still unavailable, prediction of potential AuxRE is restricted by consensus models that detect too many false positive sites. Using sequence analysis of experimentally proven AuxREs, we revealed both an extended nucleotide context pattern for AuxRE itself and three distinct types of its coupling motifs (Y-patch, AuxRE-like, and ABRE-like), which together with AuxRE may form the composite elements. Computational analysis of the genome-wide distribution of the predicted AuxREs and their impact on auxin responsive gene expression allowed us to conclude that: (1) AuxREs are enriched around the transcription start site with the maximum density in 5'UTR; (2) AuxREs mediate auxin responsive up-regulation, not down-regulation. (3) Directly oriented single AuxREs and reverse multiple AuxREs are mostly associated with auxin responsiveness. In the composite AuxRE elements associated with auxin response, ABRE-like and Y-patch are 5'-flanking or overlapping AuxRE, whereas AuxRE-like motif is 3'-flanking. The specificity in location and orientation of the coupling elements suggests them as potential binding sites for ARFs partners.

Figure 1

Context pattern of AuxRE_P&S, revealed by sequence analysis of experimentally proven AuxREs. oPWM [19] provided for mono- (A.) and dinucleotide PWMs (B.). Logos for PWMs were computed according to [60]. C. SiteGA [19] revealed significant correlations between the frequencies of locally positioned dinucleotides. The average counts of significant dependencies are shown on the Y axis per positions along AuxRE site (X axis). AuxRE core TGTCNN locates in [-3;+3] nt. D - F. Logos for potential coupling elements found on the flanks of experimentally proven AuxREs. D. ABRE-like. E. Y-patch. F. AuxRE-like.

Figure 2

Distribution of potential AuxREs in the A. thaliana genome. A. The densities of four AuxRE variants (TGTCTC, TGTCGG, TGTSTSBC and AuxRE_P&S) are shown in different genomic regions. Each density was normalized to an average density of the motif in the whole genome. B. The percentage of the Arabidopsis genes, having potential AuxREs in 2 kb and 300 bp upstream regions to the annotated TSS. C. Distribution of different AuxRE variants along the [-2000; +250] regions relative to annotated TSS. Each density on panels A and C was normalized to the average one of the respective AuxRE variant estimated for the whole genome.

Figure 3

Distribution and functional annotation of the predicted composite AuxREs/Y-patch. Panel A depicts the distribution of potential composite AuxREs relative to TSS (position +1). Panel B shows the functional annotation (GO) of the genes having the composite element AuxRE+/Y-patch+ in their regulatory regions. Functional annotation was performed using AgriGO analysis tool [26]. Annotation is represented as a graph in which each node represents a definite GO term with the value of FDR in brackets. The color of the node, from yellow to deep orange reflects the level of significance of the term which is represented by a given node. Each node is connected with the other by edges of different structure and color (annotation is given).

Figure 4

The preferences in the orientation (A) and the location (B-C) of the coupling motifs around AuxRE_P&S in the composite auxin responsive elements. A. The number of microarray experiments where the composite elements with the specific orientation of the coupling motifs were found significantly associated with auxin responsiveness (See Methods). B. The significant associations found by microarray data analysis for AuxRE+/Y-patch+, AuxRE+/ABRE-like- and AuxRE+/AuxRE-like+ composite elements, depending on the positioning of the coupling motif relative to AuxRE. The distance between the centers of the coupling motif and anchor AuxRE in nt is plotted along the x axis. The core AuxRE sequence TGTCNN locates from -3 to +3 nt. C. The conceptual summary about the composite structure of auxin responsive elements.