Yeast one-hybrid assay

The full-length CDS of OBF1 and OBF4 were cloned into the pGADT7 vector (Clontech, Beijing, China) to generate pGADT7–OBF1 and pGADT7–OBF4, respectively. The E1 segment was cloned into the pHISi-1 vector (Clontech, Beijing, China) to generate pHISi–E1. The pHisi-E1 vector was linearized with XhoI and transferred into the yeast strain YM4271, followed by incubation for 3 to 5 days at 30°C. A positive pHISi-E1 clone was used to test the proper concentration of 3-amino-1, 2, 4-triazole (3-AT) in the SD/-His medium for background expression. Selection medium containing 20 mm 3-AT was used to screen for enhancer binding proteins. pGADT7, pGADT7–OBF1 and pGADT7–OBF4 were transferred into YM4271 yeast cells containing the pHisi-E1 recombinant plasmid and grown in SD/-Leu-His supplemented with 20 mm 3-AT. After a 3 to 5–day incubation at 30°C, positive clones confirmed by PCR-sequencing were diluted 10–100 fold and spotted onto SD/-Leu-His plates containing 0, 20 and 30 mm 3-AT. The primers used in the Y1H assay are listed in S2 Table.