DNA-binding specificity and molecular functions of NAC transcription factors
Introduction
Life processes in plants depend on a multitude of transcription factors to regulate gene expression. The NAC proteins constitute one of the largest families of plant-specific transcription factors [1] and have been implicated in developmental programs, defence and abiotic stress responses (reviewed in [2]). This protein family is defined by an N-terminal domain called the NAC domain [3], which mediates DNA binding [4], [5], [6]. The three-dimensional structure of the NAC domain has been determined and shows a unique transcription factor fold [6]. However, the mode of DNA recognition is unknown.
The DNA-binding ability of NAC proteins was first suggested by the activation of the cauliflower mosaic virus (CaMV) 35S promoter [Hirt, original GenBank annotation and personal communication in 7]. Subsequently, a number of NAC proteins have been shown to bind fragments of this promoter [4], [5], [6], [8]. The identification of endogenous target genes of the NAC transcription factors has only recently been pursued. A number of genes are upregulated by overexpression of either of three stress-inducible Arabidopsis NAC genes, ANAC019, ANAC055 and ANAC072/RD26 [9], [10]. In a yeast one-hybrid screen, the three ANAC proteins bound a fragment of the early responsive to dehydration stress 1 (ERD1) promoter. Substitution analysis identified CACG (reverse complement: CGTG) as the core sequence recognized by the ANAC proteins in the ERD1 promoter [10].
To investigate the DNA binding of NAC proteins further, we performed PCR-assisted binding site selection experiments with two Arabidopsis NAC proteins, ANAC019 and ANAC092 (nomenclature from [11]). The consensus sequences obtained were evaluated by electrophoretic mobility shift assays (EMSAs), and analyses of putative NAC target gene promoters and the CaMV 35S promoter substantiated the results. NAC proteins were shown to bind DNA as dimers in an EMSA experiment with different-sized NAC proteins. A NAC domain truncation rendering the domain unable to dimerize also prevented DNA binding. Molecular functions of the NAC domain, dimerization and DNA binding, were examined using site-directed mutagenesis. The mutational analysis identified residues important for dimerization and a loop region involved in DNA binding.
Section snippets
Recombinant proteins
The Gateway system (Invitrogen) was used to obtain constructs for expression of full-length ANAC092 (At5g39610, NP_198777) as GST and MBP fusion proteins. Cloning was performed with pENTR/D Directional TOPO Cloning Kit (Invitrogen) with the following primers: 5′-CACCCTGGAAGTTCTGTTCCAGGGGCCCATGGATTACGAGGCAT-3′ and 5′-TTAGAAATTCCAAACGCAATCCAATTCTTCTGTCCCGGAC-3′. The LR Clonase Enzyme mix (Invitrogen) was used for the recombination reactions with pDEST15 (GST, Invitrogen) and with pDEST-566 (MBP,
In vitro selection of NAC binding sites
DNA sequences preferentially bound by the NAC proteins ANAC019(1–168) and ANAC092 were isolated by cyclic amplification and selection of targets (CASTing). ANAC019 and ANAC092 were chosen for the experiments because they belong to two different subgroups of NAC proteins implicated in stress response and morphogenesis, respectively [11]. Therefore, similarities between DNA sequences selected by the two proteins would likely reflect general NAC protein binding preferences. Oligonucleotides used
NAC proteins: DNA-binding specificity and dimerization
In the present study, consensus binding sites for two NAC proteins were identified using in vitro selection. EMSA investigation of NAC protein binding to the consensus sequence and to a CaMV promoter fragment substantiated the results. An EMSA experiment with two different-sized NAC proteins demonstrated that NAC proteins bind DNA as dimers. An N-terminal truncation of the NAC domain that prevents dimerization also prevented DNA binding. In addition, in silico analysis showed that potential NAC
Acknowledgements
This work was supported by grants from the Danish Research Agency (23-02-0018, 21-04-0483, 23-04-0173) (KS) and a Ph.D. stipend from the University of Copenhagen (ANO). Leila Lo Leggio and Heidi A. Ernst thank the Danish Natural Science Research Council for financial support.
References (36)
- et al.
NAC transcription factors: structurally distinct, functionally diverse
Trends Plant Sci.
(2005) - et al.
The no apical meristem gene of Petunia is required for pattern formation in embryos and flowers and is expressed at meristem and primordia boundaries
Cell
(1996) - et al.
JUMONJI, a critical factor for cardiac development, functions as a transcriptional repressor
J. Biol. Chem.
(2003) - et al.
Selection of high-affinity binding sites for sequence-specific, DNA binding proteins from random sequence oligonucleotides
Anal. Biochem.
(1995) Combinatorial gene regulation by eukaryotic transcription factors
Curr. Opin. Struct. Biol.
(1999)- et al.
Recognition of specific DNA sequences
Mol. Cell
(2001) - et al.
Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes
Science
(2000) - et al.
Genes involved in organ separation in Arabidopsis: an analysis of the cup-shaped cotyledon mutant
Plant Cell
(1997) - et al.
Arabidopsis NAC1 transduces auxin signal downstream of TIR1 to promote lateral root development
Genes Dev.
(2000) - et al.
Molecular characterization of AtNAM: a member of the Arabidopsis NAC domain superfamily
Plant Mol. Biol.
(2002)
Structure of the conserved domain of ANAC, a member of the NAC family of transcription factors
EMBO Rep.
Molecular characterization of Brassica napus NAC domain transcriptional activators induced in response to biotic and abiotic stress
Plant Mol. Biol.
A dehydration-induced NAC protein, RD26, is involved in a novel ABA-dependent stress-signaling pathway
Plant J.
Isolation and functional analysis of Arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 promoter
Plant Cell
Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsis thaliana
DNA Res.
Interactions between plant RING-H2 and plant-specific NAC (NAM/ATAF1/2/CUC2) proteins: RING-H2 molecular specificity and cellular localization
Biochem. J.
Preliminary crystallographic analysis of the NAC domain of ANAC, a member of the plant-specific NAC transcription factor family
Acta Crystallogr. D
Diverged binding specificity of Rim101p, the Candida albicans ortholog of PacC
Eukaryot. Cell
Cited by (161)
Characteristics of NAC transcription factors in Solanaceae crops and their roles in responding to abiotic and biotic stresses
2024, Biochemical and Biophysical Research CommunicationsOsNAC120 balances plant growth and drought tolerance by integrating GA and ABA signaling in rice
2024, Plant CommunicationsZFP37, C3H, NAC94, and bHLH148 transcription factors regulate cultivar-specific drought response by modulating r40C1 gene expression in rice
2023, Environmental and Experimental Botany