Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis

Abstract

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF-DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

Keywords: ARF transcription factors; auxin; plant biology; protein–DNA interaction; transcriptional regulation.

Fig. 1.

Atomic basis for high-affinity ARF-DNA binding. (A) Crystal structure of an AtARF1-DBD dimer bound to dsDNA containing two inverted TGTCGG elements (sequence below, AuxREs in bold and high-affinity residues underlined). The two monomers are colored gray and cyan, respectively, and the DNA is shown as a stick representation in orange. (B) Overlay of the ARF1-DBD [H136-G137] region and interacting nucleotides in the structures of ARF1-DBD/TGTCGG complex (AtARF1-GG, gray) and ARF1-DBD/TGTCTC complex (AtARF1-TC, green). Note that in the AtARF1-GG structure, the DNA is displaced upwards and H136 enters deeper into the major groove, allow interactions with the G₅ and G₆ bases. (C and D) Hydrogen bonding of ARF1 His136 with AuxRE nucleotides. Interaction of H136-G137 with the G₅G₆ bases in the TGTCGG-containing oligonucleotide (C) and with G₆G₇ bases of the complementary strand in TGTCTC-containing oligonucleotide (D). Note that the G₇ base is not part of the AuxRE. A backbone oxygen contributes to DNA interaction in the GG structure (C).

Fig. 2.

Cooperative action of two AuxRE motifs in an inverted repeat. (A) Genomic features of the promoter region of TMO5. (Top) Positions of AuxRE-like motifs across the promoter. The position of the two AuxRE-like motifs in an inverted repeat constellation with 7-bp spacing are indicated in red. (Bottom) Rows show AtARF5 DAP-seq peaks, DNase hypersensitive sites (DHSs), and sequence conservation among TMO5 homologs in 63 angiosperm species. (B) WT and mutated (∆1 and ∆2) sequence at 1,588–1,569 bp upstream of the start codon in the TMO5 promoter. AuxRE-like hexanucleotide motifs and the corresponding mutated nucleotides are indicated in red and blue, respectively. (C) Representative images of 5-d-old root tips that express TMO5-3xGFP driven by the WT TMO5 promoter, and ∆1 and ∆2 mutants. The roots were counterstained with propidium iodide (PI, gray). Green frames indicate the area in which GFP signals were quantified. (D) Boxplot showing the levels of fluorescent signals taken from TMO5-3xGFP driven by the WT and mutant promoter. Each dot represents the mean intensity measured from an individual root tip, and data were collected from multiple individual transgenics (SI Appendix, Fig. S3). (E) Representative images of 1-wk-old primary roots of tmo5 tmo5l1 seedlings that carry a transgene to express TMO5-tdT under the control of WT or mutant TMO5 promoter. Arrowheads indicate protoxylem strands. The roots were stained with PI. (F) Boxplots showing percentages of diarch seedlings in independent transgenic tmo5 tmo5l1 mutant seedlings carrying the transgene WT or mutated pTMO5. Each dot represents an individual transgenic line. The numbers of observed roots are shown in SI Appendix, Table S2. Asterisks in D and F indicate statistically significant differences (P value < 0.001) assessed by one-way ANOVA with post hoc Tukey test; n.s., not significant.

Fig. 3.

Genome-wide association of AuxRE repeats with auxin responsiveness. (A) Definition of DRn, IRn, and ERn of TGTCNN hexanucleotide. N indicates A, C, G, or T. n indicates the number of nucleotides between two TGTCNN half-sites (0 ≤ n ≤10). (B–D) Association of DRn-, IRn-, or ERn variants present in the upstream regions (1,500 bp; 5′UTR) of the genes with auxin responsiveness. Color saturation visualizes the significance of overrepresentation for each composite element in −log10(meta p-value) units. Auxin-activated versus -nonactivated genes (B), both up- and down-regulated versus nonresponding genes (C), and down-regulated versus noninhibited genes (D). (E–G) Significance levels for association of DR5 (E), IR8 (F), and a single TGTCGG motif (G) with up-regulation, down-regulation, and both, in −log10(meta p-value) units.

Fig. 4.

IR8 and DR5 repeats accurately predict auxin response. (A and B) The proportion of genes that showed up-regulation (red) and down-regulation (blue) by qRT-PCR within 6 h of 2,4-D treatment among genes containing an IR8- (A) or DR5-element (B) in their upstream region. Auxin responsiveness was estimated by both two-way ANOVA for a time series (P < 0.05) with one-way ANOVA with Tukey post hoc test for a time point (P < 0.05). NR, not responsive to auxin. (C–H) Time courses of the auxin response of three representative genes downstream of IR8-element: AT4G28640/IAA11, AT1G80240/DGR1, and AT4G03110/BRN1 (C–E), and three downstream of DR5-element: AT3G23030/IAA2, AT1G19220/ARF19, and AT3G54000/TIP41-like (F–H). The ratio of expression levels in auxin-treated seedlings to that in mock sample at four time points: 15 min, 1 h, 2 h, 6 h are shown as mean ± SD. Asterisks indicate significant differences by one-way ANOVA with Tukey post hoc test (*<0.05, **<0.01, ***<0.001).

Fig. 5.

An IR8 element in the IAA11 promoter functions as a composite AuxRE. (A) (Top) Positions of AuxRE-like motifs across the IAA11 promoter. The position of the two AuxRE-like motifs in an inverted repeat constellation with 7-bp spacing are indicated in red. (Bottom) Rows show AtARF5 DAP-seq peaks, DHSs, and sequence conservation among IAA11 homologs in 63 angiosperm species. (B) Sequence of the WT and mutant IR8 element in the IAA11 promoter (C) Boxplot compares the intensity levels from n3GFP driven by pIAA11WT (red), pIAA11∆1 (green), and pIAA11∆2 (blue) in vascular cells in the transition zone of primary roots grown without exogenous auxin (control condition). Each dot indicates the mean intensity over an area of same size measured for an individual root, and data were collected from multiple individual transgenics. (D) Auxin response of the WT and mutated IAA11 promoters based on the signals from n3GFP coupled to the promoters. The boxplot indicates the ratio between the signal levels in vascular cells in the elongation zone of roots treated with auxin for 6 h and that of roots under control condition. Each dot indicates the ratio estimated for individual root relative to the normalized control level for specific line. Asterisks in C and D indicate statistically significant differences (P < 0.001 by one-way ANOVA with post hoc Tukey test). n.s: not significant.

Fig. 6.

Single-molecule FRET assays reveal differential ARF-DNA affinities. (A) Cartoon describing the single-molecule FRET assay. IR8- or DR5-containing (arrows) ds oligonucleotide carrying two FRET-compatible dyes (magenta and green stars) are immobilized on a coverslip. Arrows indicate position of TGTCGG sequences. Binding of ARF proteins (dimer indicated in orange/blue) to the oligo leads to bending of DNA bending and slight displacement of the dyes thereby decreasing FRET efficiency. (B) Titrations of ARF1-DBD and ARF5-DBD proteins (concentration in nM on x-axis) on surface-immobilized DR5 or IR8 oligonucleotides. The y-axis shows the fraction of DNA bound to protein derived from FRET efficiency distribution. Dissociation constants (K_d) are given in the legend, with their 95% intervals of confidence from the fit.