|
 Curently online :
28 |
|
 Total visitors :
7671891 |
|
|
Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis
Friday, 2020/10/02 | 06:21:32
|
|
Alejandra Freire-Rios, Keita Tanaka, Isidro Crespo, Elmar van der Wijk, Yana Sizentsova, Victor Levitsky, Simon Lindhoud, Mattia Fontana, Johannes Hohlbein, D. Roeland Boer, Victoria Mironova, and Dolf Weijers PNAS September 29, 2020 117 (39) 24557-24566. SignificanceThe plant hormone auxin controls many aspects of growth and development. It does so by changing the activity of AUXIN RESPONSE FACTOR (ARF) proteins that recognize specific DNA elements in plant genes and switch gene expression on or off. A major question in plant biology is how these ARF proteins bind unique DNA sequences and thus select which genes are regulated by auxin. Here, the authors systematically study the DNA architecture required for high-affinity binding of ARF proteins, and for auxin-dependent gene regulation in the plant Arabidopsis thaliana. This work shows that repeats of ARF recognition sites are critical for function, and reveals the molecular basis for the distinct activities of different repeat structures. AbstractThe 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.
See: https://www.pnas.org/content/117/39/24557
Figure 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. |
Back
Print
View: 323
|
|
[ Other News ]___________________________________________________
|
(95).png "Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis")
