Fig. 4: Mapping and functional cooperation of PIF4 and CDF2 binding in the YUCCA8 promoter in vivo and overall structure modelling of the CDF2^DOF–DNA complex.

a, Windows for Assay for Transposase-Accessible Chromatin using sequencing (ATAC–seq) analysis³¹ and CDF2 and PIF4²¹ binding profile to the YUCCA8 gene. The DOF-binding sites and G-boxes are shown through the whole gene body. The locations of amplicons for ChIP–qPCR analysis in b and probes for EMSA experiments in f and g are also shown. b, ChIP–qPCR analysis of RNAPII enrichment in the transcribed regions of YUCCA8 in transgenic plants carrying CDF2::HA-CDF2 in cdf2 and cdf2 pif4 backgrounds. The transcribed region of YUCCA8 is marked with the black box that matches with positions of the analyzed amplicons in a. Data are represented as means ± SEM of three independent amplifications. Statistical significance was determined by pairwise one-sided t-test (for amplicons 1-8 in CDF2::HA-CDF2; cdf2 versus CDF2::HA-CDF2; cdf2 pif4, P = 0.396, 0.112, 0.306, 0.047, 0.017, 0.024, 0.156 and 0.03). Asterisks mark significant differences, ^∗P < 0.05. c, RT–qPCR analysis of YUCCA8 mRNA levels in Col-0 WT (wild type) and transgenic plants carrying CDF2::HA-CDF2 in cdf2 and cdf2 pif4 backgrounds. Data are represented as means ± SEM of three independent amplifications. All values are normalized to APA1 levels. Statistical significance was determined by pairwise one-sided t-test (Col-0 versus CDF2::HA-CDF2; cdf2, P = 0.0328085, Col-0 versus CDF2::HA-CDF2; cdf2 pif4, P = 0.0010115 and CDF2::HA-CDF2; cdf2 versus CDF2::HA-CDF2; cdf2 pif4, P = 0.0004875). Asterisks mark significant differences, ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001. d, Modelled structure of the CDF2^DOF–DNA complex. e, DOF domain architecture of CDF2 and alignment of the DOF domain among CDF1, 2, 3, 5 and PEAR1, DAG1 and SCAP1 proteins in Arabidopsis. f, Overview of the long WT and mutant DNA probes (95 bp) for EMSA experiments for analysis of CDF2^DOF DNA-binding specificity and affinity. g, Interactions between CDF2^DOF protein and DNA probes analyzed by EMSA. h,i, Overview of short WT (h) and mutant DNA probes (38 bp) (i) within the long YUCCA8 DNA probe, and interactions between CDF2^DOF and DNA probes analyzed by EMSA. j, Mutations in the predicted residues of the CDF2^DOF domain for DNA binding. Alignment among some Arabidopsis DOF TFs is shown in e. Interactions between DNA and CDF2^DOF WT and mutant proteins in the predicted residues of CDF2^DOF domain analyzed by EMSA. EMSA assays in g, i and j were performed three times with similar results.

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