小背景：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.

前期基础：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.

提出问题：However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive.

主要研究：Here we biochemically and functionally interrogate modes of ARF–DNA interaction.

结果1：We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites.

结果2：We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo.

结果3：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.

结果4：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.

结论：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.