title: A high-throughput screening system for Arabidopsis transcription factors and its application to Med25-dependent transcriptional regulation.
abstract: The activities of transcription factors (TFs) require interactions with specific DNA sequences and other regulatory proteins. To detect such interactions in Arabidopsis, we developed a high-throughput screening system with a Gateway-compatible Gal4-AD-TF library of 1589 Arabidopsis TFs, which can be easily screened by mating-based yeast-one-hybrid (Y1H) and yeast-two-hybrid (Y2H) methods. The efficiency of the system was validated by examining two well-characterized TF-DNA and TF-protein interactions: the CHE-CCA1 promoter interaction by Y1H and NPR1-TGAs interactions by Y2H. We used this system to identify eight TFs that interact with a Mediator subunit, Med25, a key regulator in JA signaling. We identified five TFs that interacted with the GCC-box cis-element in the promoter of PDF1.2, a downstream gene of Med25. We found that three of these TFs, all from the AP2-EREBP family, interact directly both with Med25 and the GCC-box of PDF1.2, suggesting that Med25 regulates PDF1.2 expression through these three TFs. These results demonstrate that this high-throughput Y1H/Y2H screening system is an efficient tool for studying transcriptional regulation networks in Arabidopsis. This system will be available for other Arabidopsis researchers, and thus it provides a vital resource for the Arabidopsis community.
authors.affiliation: National Laboratory for Protein Engineering and Plant Genetic Engineering, College of Life Sciences, Peking University, Beijing 100871, People's Republic of China.
authors.forename: Bin
authors.initials: B
authors.lastname: Ou
authors.forename: Kang-Quan
authors.initials: KQ
authors.lastname: Yin
authors.forename: Sai-Nan
authors.initials: SN
authors.lastname: Liu
authors.forename: Yan
authors.initials: Y
authors.lastname: Yang
authors.forename: Tren
authors.initials: T
authors.lastname: Gu
authors.forename: Jennifer Man
authors.initials: JM
authors.lastname: Wing Hui
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.forename: Jin
authors.initials: J
authors.lastname: Miao
authors.forename: Youichi
authors.initials: Y
authors.lastname: Kondou
authors.forename: Minami
authors.initials: M
authors.lastname: Matsui
authors.forename: Hong-Ya
authors.initials: HY
authors.lastname: Gu
authors.forename: Li-Jia
authors.initials: LJ
authors.lastname: Qu
mesh_terms: D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000378:metabolism*; D004268:DNA-Binding Proteins; D018506:Gene Expression Regulation, Plant; D015723:Gene Library; D057166:High-Throughput Screening Assays / Q000379:methods*; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011485:Protein Binding; D015203:Reproducibility of Results; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D029681:Arabidopsis Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C475686:PFT1 protein, Arabidopsis; D014157:Transcription Factors