부산대학교 도서관

Fine-tuning of nutrient uptake and response is indispensable for maintenance of nutrient homeostasis in plants, but the details of underlying mechanisms remain to be elucidated. NITRATE-INDUCIBLE GARP-TYPE TRANSCRIPTIONAL REPRESSOR 1 (NIGT1) family proteins are plant-specific transcriptional repressors that function as an important hub in the nutrient signaling network associated with the acquisition and use of nitrogen and phosphorus. Here, by yeast two-hybrid assays, bimolecular fluorescence complementation assays, and biochemical analysis with recombinant proteins, we show that Arabidopsis NIGT1 family proteins form a dimer via the interaction mediated by a coiled-coil domain (CCD) in their N-terminal regions. Electrophoretic mobility shift assays defined that the NIGT1 dimer binds to two different motifs, 5'-GAATATTC-3' and 5'-GATTC-N38-GAATC-3', in target gene promoters. Unlike the dimer of wild-type NIGT1 family proteins, a mutant variant that could not dimerize due to amino acid substitutions within the CCD had lower specificity and affinity to DNA, thereby losing the ability to precisely regulate the expression of target genes. Thus, expressing the wild-type and mutant NIGT1 proteins in the nigt1 quadruple mutant differently modified NIGT1-regulated gene expression and responses towards nitrate and phosphate. These results suggest that the CCD-mediated dimerization confers dual mode DNA recognition to NIGT1 family proteins, which is necessary to make proper controls of their target genes and nutrient responses. Intriguingly, two 5'-GATTC-3' sequences are present in face-to-face orientation within the 5'-GATTC-N38-GAATC-3' sequence or its complementary one, while two 5'-ATTC-3' sequences are present in back-to-back orientation within the 5'-GAATATTC-3' or its complementary one. This finding suggests a unique mode of DNA binding by NIGT1 family proteins and may provide a hint as to why target sequences for some transcription factors cannot be clearly determined. Author summary: Expression of genes is tightly regulated by transcription factors that physically interact with DNA. Transcription factors recognize specific DNA sequences and selectively regulate the expression of target genes through the specific interactions. We previously found that a group of plant-specific transcription factors, NIGT1 family proteins, recognize two different types of DNA sequences to modulate the expression of a number of genes associated with uptake of nitrogen and phosphorus or related metabolism in Arabidopsis. However, it was unknown how NIGT1 family proteins recognize different types of sequences. In this study, we reveal that dimerization of NIGT1 family proteins is essential to the dual specificity of NIGT1 family proteins and therefore loss of the dimerization ability alters specificity and affinity of NIGT1 family proteins to target DNA sequences. We further suggest that the dimerization of NIGT1 family proteins is an evolutionarily conserved feature to properly respond to changes in nutrient availability. These findings would advance our knowledge on the mode of DNA recognition by transcription factors and have further implications for structural basis on the diverse patterns of the interaction between DNA and transcription factors. [ABSTRACT FROM AUTHOR]