Capturing Auxin Response Factors Syntax Using DNA Binding Models

Abstract

Auxin is a key hormone performing a wealth of functions throughout the life cycle of plants. It acts largely by regulating genes at the transcriptional level through a family of transcription factors called auxin response factors (ARFs). Even though all ARF monomers analyzed so far bind a similar DNA sequence, there is evidence that ARFs differ in their target genomic regions and regulated genes. Here, we report the use of position weight matrices (PWMs) to model ARF DNA binding specificity based on published DNA affinity purification sequencing (DAP-seq) data. We found that the genome binding of two ARFs (ARF2 and ARF5/ Monopteros [MP]) differ largely because these two factors have different preferred ARF binding site (ARFbs) arrangements (orientation and spacing). We illustrated why PWMs are more versatile to reliably identify ARFbs than the widely used consensus sequences and demonstrated their power with biochemical experiments in the identification of the regulatory regions of IAA19, an well-characterized auxin-responsive gene. Finally, we combined gene regulation by auxin with ARF-bound regions and identified specific ARFbs configurations that are over-represented in auxin-upregulated genes, thus deciphering the ARFbs syntax functional for regulation. Our study provides a general method to exploit the potential of genome-wide DNA binding assays and to decode gene regulation.

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

“…Given that TGTC-based hexamers are abundant in plant genomes, and cannot by themselves explain or help predicting auxin response confidently (37), it is likely that a composite element is involved in auxin-dependent gene regulation at genome-wide scale. Earlier enrichment of particular AuxRE compositions, including inverted repeats spaced by seven or eight bases, have been shown in the promoters of auxin-responsive genes and in ARF5 DAP-Seq regions (50). We systematically addressed if the occurrence of spaced repeats of AuxREs in gene upstream regions is associated with auxin responsiveness of gene expression in individual transcriptomes.…”

Deep conservation of cis-element variants regulating plant hormonal responses

“…To correct for both these factors, background rates were determined empirically by calculating the conservation score distribution for the two variant nucleotides 8 bp upstream to the core sequence (NNnnnnnnTGTC for the auxRE; Supplemental Figure 1B). auxREs are sometimes found in close proximity to one another (O'Malley et al, 2016;Stigliani et al, 2018), which could affect the distribution of nucleotides at these positions. However, the overall distribution of dinucleotides at position 28 to the auxRE was found to be similar to the overall distribution in promoters, indicating that, genome wide, this effect is negligible (Supplemental Figure 1B).…”

“…To test the impact of the motif variants in native promoters, we modified the conserved TGTCCC auxRE motif to the weakresponse variant TGTCAC in the promoters of two auxinresponsive genes, IAA2 and IAA4. Both promoters had a second TGTCCC site with a 5-bp spacer, a configuration preferred by ARF dimers (O'Malley et al, 2016;Stigliani et al, 2018). As this pair arrangement was perfectly conserved within the Brassicaceae, we mutated both motifs ( Figures 7A to 7F).…”

Deep conservation of response element variants regulating plant hormonal responses

“…To test the impact of the motif variants in native promoters, we modified the conserved TGTC CC auxRE motif to the weak-response variant TGTC AC in the promoters of IAA2 and IAA4 . Both promoters had a second TGTC CC site with a 5-bp spacer, a configuration preferred by ARF dimers 44,45 . As this pair arrangement was perfectly conserved within the Brassicacea, we mutated both motifs (Fig.…”