Extended Data Fig. 3: Characterization of the TFs and TF binding sites that regulate class A ARF expression.

a, Yeast one-hybrid promoter–transcription factor interaction network for class A ARF genes. Green boxes correspond to the class A ARF; pink boxes are transcription factors binding to the ARF promoters. TF-associated functions and expression analysis are indicated in the upper and lower small boxes and colour-coded as indicated in the key. Note that when two promoter fragments were used for the screen (see Methods), 35 out of 36 regulators bound to the more proximal fragment, supporting previous observations that the majority of transcription factor binding sites reside within a few kb of the transcriptional start site⁵⁵. b, Frequency of TF gene families in the Y1H library collection (black) and in the Y1H network (white). Only families represented by at least two members in the Y1H network were analysed. The network is overrepresented with members of the WRKY and SPL TF families. Statistical analysis: hypergeometric test significant to 5% (*; P = 4e-05 for WRKY family and P = 0.044 for SPL family). Sample sizes for TFs in Y1H library in black/Y1H network in white: n = 29/8 TFs (WRKY); n = 68/6 (ZFP); n = 91/6 (AP2/ERF); n = 44/2 (NAC); n = 7/2 TFs (SPL); n = 52/2 TFs (homeobox); n = 61/2 TFs (bHLH). c, TF expression in the RAM³⁸ and the SAM^39,40. 50% of the identified TFs are expressed in both shoots and roots, whereas 24% and 14% are expressed specifically in roots or shoots respectively. d, Known functions of the TFs in the Y1H network based on a literature search (see also Supplementary Table 2). e, Boxplot representation of the distribution of class A ARF promoter ranks. For TFs with established binding models, we ranked class A ARF promoters among all Arabidopsis promoters based on the score of the predicted TF binding sites. We repeated the same operation with a set of randomly chosen TFs from different families (see Methods). The comparison of rank distributions with those of a set of randomly chosen TFs from different families revealed significantly higher ranks for eY1H-identified TFs (see also Supplementary Table 3). Statistical analysis: one-sided t-test. Sample sizes: n = 29 for eY1H-selected TFs and n = 100 for randomly selected TFs. Data are represented as boxplots where the middle line is the median, the lower and upper hinges correspond to the first and third quartiles, the upper whisker extends from the hinge to the largest value no further than 1.5× interquartile range (IQR) from the hinge and the lower whisker extends from the hinge to the smallest value at most 1.5× IQR of the hinge. All the individual values are plotted. f, Summary of the DAP-seq analysis for the 17 TFs (see also Supplementary Table 3). g, Example of DAP-seq data, here a DAP-seq peak for WRKY33 in the promoter of ARF8. DAP-seq (f, g) thus confirms experimentally inferred bindings (e) for 4 of the 17 (24%) TFs for which DAP-seq data are available (see also Supplementary Table 3). Note also that chromatin immunoprecipitation sequencing (ChIP-seq) confirms the binding of WUSCHEL to the ARF8 promoter²¹.