Structural Basis for DNA Binding Specificity by the Auxin-Dependent ARF Transcription Factors

Abstract

Auxin regulates numerous plant developmental processes by controlling gene expression via a family of functionally distinct DNA-binding auxin response factors (ARFs), yet the mechanistic basis for generating specificity in auxin response is unknown. Here, we address this question by solving high-resolution crystal structures of the pivotal Arabidopsis developmental regulator ARF5/MONOPTEROS (MP), its divergent paralog ARF1, and a complex of ARF1 and a generic auxin response DNA element (AuxRE). We show that ARF DNA-binding domains also homodimerize to generate cooperative DNA binding, which is critical for in vivo ARF5/MP function. Strikingly, DNA-contacting residues are conserved between ARFs, and we discover that monomers have the same intrinsic specificity. ARF1 and ARF5 homodimers, however, differ in spacing tolerated between binding sites. Our data identify the DNA-binding domain as an ARF dimerization domain, suggest that ARF dimers bind complex sites as molecular calipers with ARF-specific spacing preference, and provide an atomic-scale mechanistic model for specificity in auxin response.

Cistrome and Epicistrome Features Shape the Regulatory DNA Landscape

“…For ER-TGTC, we observed three spacing groups at 4-8, 15-18, and 25-28 bp. Importantly, our results exactly matched the known spacing of 10-12 bp for DR (Ulmasov et al, 1997) and 15-18 bp for ER (Boer et al, 2014). We also identified novel binding events at IR repeats, which showed similar spacing preferences to those seen for ER-TGTC repeats but had only two spacing groups (15-16 and 25-27 bp).…”

“…Several TFs were at the opposite extreme, where strong DAP-seq peaks contained much less than one motif on average (STZ, NAP, ARF2, and MP/ARF5; Figure S5B). Unexpectedly, this group included two ARFs (ARF2 and MP/ARF5) with only 0.1-0.2 motifs per peak despite evidence that they bind to motif repeats in vitro and in vivo (Boer et al, 2014;Ulmasov et al, 1997). This suggests that direct examination is needed to understand the more complex binding site architecture required for strong binding for some families.…”

“…The spacing between individual AuxREs is important as the DR was bound only when tested with spacing of 10-12 bp (i.e., between the two T's in bold TGTCTC-N 4-6 -TGTCTC), and the ER AuxRE with spacing of 15-18 bp (TGTCGG-N 6-9 -CCGACA) (Ulmasov et al, 1997). A crystal structure of an ARF bound to an ER AuxRE showed that the 15-18 bp spacing allowed the bound ARF homodimer to stabilize through interaction of the ARF dimerization domain (DD) (Boer et al, 2014). Similarly, the DR spacing preference may be explained by stabilizing interactions through a second dimerization domain, the III/IV domain ( Figure 6B) (Nanao et al, 2014).…”

“…See also Figure S3 and Table S1. (Nanao et al, 2014) and at an ER by the dimerization domain (bottom) (Boer et al, 2014). (C) Relative frequencies of DAP-seq peaks at DR, ER, and IR pairs for Arabidopsis (AtARF5 and AtARF2) and maize (ARF5/ZmARF29) proteins interrogating Arabidopsis (At-gDNA) or maize (Zm-gDNA) DAP-seq libraries.…”

Auxin-Induced Modulation of ETTIN Activity Orchestrates Gene Expression in Arabidopsis

“…The Arabidopsis thaliana genome encodes 23 ARFs and the N-terminal region of each ARF possesses a conserved DNA binding domain (DBD) able to mediate ARF dimerization and recognize auxin response elements (AuxREs) with the general sequence motif TGTCNN. Individual ARFs have certain degrees of specificity and preference for both sequence and motif spacing (Boer et al, 2014). In the C-terminal region, all ARFs, except ARF3, ARF13, and ARF17, contain the conserved PB1 (Phox/Bem1p) domain through which they interact with Aux/IAA proteins (Boer et al, 2014;Guilfoyle, 2015;Korasick et al, 2015;Salehin et al, 2015;Chandler, 2016).…”