RRC ID 65947
Author Tamura T, Endo H, Suzuki A, Sato Y, Kato K, Ohtani M, Yamaguchi M, Demura T.
Title Affinity-based high-resolution analysis of DNA binding by VASCULAR-RELATED NAC-DOMAIN7 via fluorescence correlation spectroscopy.
Journal Plant J
Abstract VASCULAR-RELATED NAC-DOMAIN7 (VND7) is the master transcription factor for vessel element differentiation in Arabidopsis thaliana. To identify the cis-acting sequence(s) bound by VND7, we employed fluorescence correlation spectroscopy (FCS) to find VND7-DNA interactions quantitatively. This identified an 18-bp sequence from the promoter of XYLEM CYSTEINE PEPTIDASE1 (XCP1), a direct target of VND7. A quantitative assay for binding affinity between VND7 and the 18-bp sequence revealed the core nucleotides contributing to specific binding between VND7 and the 18-bp sequence. Moreover, by combining the systematic evolution of ligands by exponential enrichment (SELEX) technique with known consensus sequences, we defined a motif termed the Ideal Core Structure for binding by VND7 (ICSV). We also used FCS to search for VND7 binding sequences in the promoter regions of other direct targets. Taking these data together, we proposed that VND7 preferentially binds to the ICSV sequence. Additionally, we found that substitutions among the core nucleotides affected transcriptional regulation by VND7 in vivo, indicating that the core nucleotides contribute to vessel-element-specific gene expression. Furthermore, our results demonstrate that FCS is a powerful tool for unveiling the DNA-binding properties of transcription factors.
Volume 100(2)
Pages 298-313
Published 2019-10-1
DOI 10.1111/tpj.14443
PMID 31313414
MeSH Arabidopsis / genetics Arabidopsis / metabolism* Arabidopsis Proteins / genetics Arabidopsis Proteins / metabolism* Gene Expression Regulation, Plant / genetics Gene Expression Regulation, Plant / physiology Plants, Genetically Modified / genetics Plants, Genetically Modified / metabolism Promoter Regions, Genetic / genetics SELEX Aptamer Technique Spectrometry, Fluorescence Transcription Factors / genetics Transcription Factors / metabolism*
IF 6.141
Arabidopsis / Cultured plant cells, genes rpc00008