Fig. 7

CmNF-YB8 binds to the promoter of cmo-MIRNA156. a Schematic representation of the upstream region of the foldback structure of the cmo-miR156 precursors a and b (pre-miR156a and pre-miR156b, respectively; green boxes). The blue box indicates the promoter of the cmo-MIR156 gene. Asterisks correspond to putative CCAAT boxes and lines above the blue box indicate the fragments amplified in the ChIP-PCR analysis. P0: +175 ~ +321 bp, P1: −266 ~ −498 bp, P2: −473 ~ −720 bp, P3: −701 ~ −914 bp, P4: −893 ~ −1086 bp, P5: −1121 ~ −1318 bp, P6: −1289 ~ −1621 bp, P7: −1599 ~ −1754 bp relative to the major transcription start site (TSS) of cmo-MIR156. Lines below the blue box indicate the fragments used in the yeast one-hybrid analysis shown in d. P8: −1421 ~ −1754 bp, P9: −1083 ~ −1436 bp, P10: −610 ~ −1082 bp, P11: −263 ~ −501 bp. b ChIP-enrichment of the indicated fragments (P0–P7) in the cmo-MIR156 promoter. Chromatin from 35S:CmNF-YB8-GFP chrysanthemum was immuno-precipitated with an anti-GFP antibody, and the amount of the indicated DNA was determined by qPCR. A pre-cmo-miR156a fragment (P0) was amplified as a negative control. c The P9 wild-type cis-element (CAAT box) and its nucleotide substitutions in the mutant version (P9m) are underlined. d Analysis of CmNF-YB8 binding to the promoter of cmo-MIR156 in a yeast one-hybrid system. The empty prey vector (AD) was used as a negative control. Interactions between bait and prey were determined by cell growth on synthetic dropout nutrient medium lacking Trp, Leu, and His, and containing 3 mM 3-amino-1,2,4-triazole (3-AT). e Interaction between CmNF-YB8 and cmo-MIR156 promoter using a dual-luciferase reporter assay in Nicotiana benthamiana leaves. A cmo-MIR156 promoter fragment: 0 ~ −1436 bp (from P9 to TSS) was used in this assay. mPro-miR156 is the same promoter fragment with a mutated CAAT cis-element (P9m). LUC vectors contain the REN gene under the control of the 35S promoter as a positive control. Samples were infiltrated into N. benthamiana leaves, and LUC and REN activities were assayed 3 days after infiltration. The ratio of LUC/REN of the empty vector (SK) co-transformed with LUC empty vector was used as calibrator (set as 1). Three independent experiments were performed and error bars indicate standard deviation