The interaction between MYB proteins and their target DNA binding sites

Biochimica et Biophysica Acta 1819 (2012) 67–77

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Review

The interaction between MYB proteins and their target DNA binding sites Michael B. Prouse a, Malcolm M. Campbell a, b,⁎ a b

Centre for the Analysis of Genome Evolution & Function, Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St., Toronto, ON, Canada M5S 3B2 Department of Biological Sciences, University of Toronto Scarborough, 1265 Military Trail, Toronto ON, Canada M1C 1A4

a r t i c l e

i n f o

Article history: Received 20 June 2011 Received in revised form 17 October 2011 Accepted 18 October 2011 Available online 28 October 2011 Keywords: MYB Transcription factor DNA binding sites Transcriptional regulation Molecular modelling

a b s t r a c t Members of the MYB family of transcription factors are found in all eukaryotic lineages, where they function to regulate either fundamental cellular processes, or specific facets of metabolism or cellular differentiation. MYB transcription factors regulate these processes through modulation of transcription at target genes, to which they bind in a sequence-specific manner. Over the past decades, insights have been gained into the molecular interactions between MYB proteins and their cognate DNA targets. This review focuses on those insights, the emergence of common themes in DNA binding by diverse MYB family members. The review also considers gaps in the current knowledge of MYB–DNA interactions, particularly for plant MYB proteins, and how emerging techniques that examine protein–DNA interactions can fill these gaps. © 2011 Elsevier B.V. All rights reserved.

1. Introduction Growth and development are orchestrated by the activity of sequence-specific transcription factors, proteins that function to reconfigure gene expression in response to external and internal cues. Sequence-specific transcription factors can act as transcriptional activators, repressors, or both [1]. Sequence-specific transcription factors frequently have a modular structure — comprising a DNAbinding domain together with a transcriptional regulatory domain [2]. Based on the similarities of the DNA-binding domain, transcription factors have been categorised into families or superfamilies, with several such groups composed of one hundred or more

Abbreviations: R, MYB repeat; AMV, avian myeloblastosis virus; C1, COLORED1 locus; MBS, MYB binding site; EMSA, electrophoretic mobility-shift assay; AGRIS, Arabidopsis gene regulatory information server; PLACE, Plant cis-acting regulatory elements database; TRANSFAC, Transcription factor database; UTR, untranslated regions; ChIP-chip, chromatin immunoprecipitation on chip; ChIP-seq, chromatin immunoprecipitation followed by high throughput sequencing; PDB, Protein data bank; TRFL, TRF1/2Like genes; IBP, indicator binding protein group; SMH, single MYB histone group; WER, WEREWOLF; Kd, dissociation constant; MSA, M phase-specific activator element; TF, transcription factor; SNP, sodium nitroprusside; GSNO, S-nitrosoglutathione; CAST, cyclic amplification and selection of targets; SELEX, systematic evolution of ligands by exponential enrichment; FLP, FOUR LIPS; GL3, GLABRA3; GL1, GLABRA1; bHTH, basic helix–turn–helix; GR, glucocorticoid receptor; CPC, CAPRICE; WBS, WER-binding site; IFN-g, human interferon-g; LCR, locus control region ⁎ Corresponding author at: Department of Cell & Systems Biology, University of Toronto, 25 Willcocks St., Toronto, ON, Canada M5S 3B2. Tel.: + 1 416 946 0817; fax: + 1 416 978 5878. E-mail addresses: [email protected] (M.B. Prouse), [email protected] (M.M. Campbell). 1874-9399/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.bbagrm.2011.10.010

members [3,4]. The MYB superfamily is one of the largest and most diverse families of sequence-specific transcription factors [5,6]. In animals, the MYB superfamily is relatively small, generally comprising four or five proteins [6-9]. Animal MYB superfamily members regulate gene expression related to cell division or a discrete subset of cellular differentiation events [10-12]. By contrast, the MYB superfamily in plants has expanded dramatically, with 100–200 MYB family members commonly found in individual plant species [13]. In plants, MYB proteins regulate a vast array of metabolic, cellular and developmental processes [13-15]. Much is known about the specifics of the interaction between animal MYB proteins and their cognate DNA binding sites. By contrast, the knowledge of the details of MYB–DNA interactions in plants is rather incomplete. In this review we consider the current state of knowledge with respect to MYB–DNA interactions in animals, and contrast this with what is known in plants, suggesting means by which the gap in knowledge in plants can be addressed. 2. The nature of MYB proteins 2.1. The MYB transcription factor superfamily The MYB superfamily is found in all major eukaryotic lineages, and is thought to be more than 1 billion years old [6,8,16,17]. MYB proteins acquired their name from v-MYB, the oncogenic component of avian myeloblastosis virus (AMV), where the sequence-specific MYB domain was initially discovered [18]. The cellular counterpart of v-MYB is cMYB, a three repeat (R1R2R3) MYB protein that plays a critical role in controlling the proliferation and differentiation of hematopoietic cells [19]. c-MYB mutations that alter target gene expression drastically

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reduce the proliferation of hematopoietic cells [20]. In keeping with this, homozygous c-MYB knock-out lines of mice die before reaching day 15 of the foetal lifecycle due to the inability to sustain hepatic erythropoiesis [19]. MYB superfamily members are characterised by a highly conserved DNA-binding domain, referred to as the MYB domain, which generally consists of up to three imperfect amino acid repeats (R1, R2, and R3) of 50–53 amino acids [6]; however, there are certain MYB proteins that do not follow this trend, possessing at least four MYB repeats [9]. Each of the MYB repeats, within the MYB domain, gives rise to a helix–turn–helix secondary structure. The MYB domain is predominantly found within the N-terminus of MYB-proteins [21]; however, MYB domains recently have also been discovered within the C-termini of MYB-proteins [22]. Each MYB repeat consists of several highly conserved tryptophan residues that are regularly spaced forming a hydrophobic core [23]. In contrast to MYB domain, the C-terminal region of MYB proteins is characteristically highly variable from one MYB protein to another, and usually functions as either an activation or repression domain [14,16,21,24]. This gives rise to a wide range of variability both structurally and functionally within the MYB superfamily. 2.2. Animal MYB proteins As is the case with c-MYB, animal MYB superfamily members possess three MYB-repeat proteins [23,25,26]; although, there are some notable exceptions that deviate from this, including human SNAPc 190 and TRF1 [7,9]. In all annotated vertebrate genomes, there are only three MYB proteins with three MYB-repeats: A-MYB, B-MYB, and c-MYB [6,8]. A-MYB and B-MYB proteins are R1R2R3–MYB nuclear transcription factors expressed in hematopoietic cells, epithelial cells, and fibroblasts [27]. A-MYB negatively regulates cellular proliferation [11], while B-MYB positively regulates cell growth control, differentiation, and cancer [28]. 2.3. Plant MYB proteins In comparison to animals, the MYB superfamily is greatly expanded in plants [17,21,24]. For example, of the over 1600 sequence-specific transcription factors identified in the genome of the model dicotyledenous plant, Arabidopsis thaliana, almost 10% are members of the MYB transcription factor family [5,13]. In contrast to animals, A. thaliana has 5 three-repeat MYB proteins, and 126 two-repeat (R2R3) MYB proteins, [4,5,13,15,21,29], while the monocotyledon plant rice (Oryza sativa) has over 110 predicted R2R3–MYB proteins [4] (http:// grassius.org/tf_family.html?KEYWORDS=MYB&SPECIES=2). In addition, single-repeat MYB proteins have been identified in plants and animals in increasing numbers [30-57]. Although, single-repeat MYB proteins have been identified in both animals and plants, the majority of single-repeat MYB proteins have been characterised in plants. As their name implies, R2R3–MYB proteins have two MYB repeats [21]. R2R3–MYB proteins comprise the largest group of MYB transcription factors in the MYB superfamily and appear to be specific to plants [13]. Plant R2R3–MYB proteins regulate a myriad of processes, including primary and secondary metabolism; regulation of cell fate and identity; regulation of plant development; and responses to biotic and abiotic stresses [13-15,24,58-64]. While analogous processes, such as regulation of cell fate and identity, can be found in animals, the precise functions associated with R2R3–MYB proteins appear to be plant specific [13-15]. 2.4. Single MYB-repeat proteins Single MYB-repeat proteins can be classified into the following two groups: 1) Indicator Binding Protein (IBP) group, and 2) Single MYB Histone (SMH) group. The IBP group of proteins includes

RTBP1 from rice, AtTRP1 and AtTBP1 from A. thaliana [7,33,38], as well as the highly characterised telomeric DNA-binding proteins TRF1, TRF2, RAP1 and Taz1. SMH proteins are a novel group of single MYBrepeat proteins that have only been identified in plants. SMH group of proteins include PcMYB1 from Petroselinum crispum, AtTRB1, AtTRB2, AtTRB3 from Arabidopsis, and Smh1 from Maize. AtTRB1, AtTRB2, AtTRB3 have been studied in detail, all sharing a single MYB-repeat more similar to R2 than R1 and R3 [48]. In A. thaliana, single-repeat MYB proteins CAPRICE (CPC), TRYPTICHON (TRY), ETC1 (ENHANCER OF TRY and CPC) and ETC2 have been identified [65,66]. 2.5. Expansion and diversification of the MYB family Two theories of how the MYB superfamily evolved have been constructed based on parsimony [8]. The first is formulated on the premise that three-repeat MYB proteins are closely related to vertebrate c-MYB and other similar three-repeat MYB proteins in other eukaryotic groups, such as ciliates and slime molds [67,68]. These primitive proteins are predicted to have existed before the divergence between animals and plants [68]. This theory proposes that R2R3–MYB proteins originated recently from three-repeat MYB proteins due to loss of R1-MYB repeat [67,69]. The second theory postulates that within an ancient R2R3 predecessor that there was a domain duplication and subsequent gain of R1, suggesting that R2R3 is a precursor of MYB3R [70]. Common to both theories, there was a vast expansion of R2R3–MYB proteins in plants via duplications of entire genes [8]; however, the expansion was restricted for the three-repeat MYB proteins in both animals and plants. Comparisons of DNA-binding specificities and functional roles between MYB proteins with different repeats could help elucidate the nature of the evolutionary pathway for MYB proteins. 3. DNA targets of MYB family members 3.1. Animal MYB DNA-binding sites The DNA target of animal three-repeat MYB transcription factors was first determined by isolation of chicken genomic DNA fragments bound by v-MYB on filters [10] and by comparison of putative MYB binding sites within the SV40 enhancer region [71]. Binding-site selection methods with c-MYB protein resulted in added minor extensions to the c-MYB consensus sequence. The c-MYB consensus sequence was found to be ((T/C)AAC(G/T)G(A/C/T)(A/C/T)) and was termed MYB binding site I (MBSI) [25,72]. Mutational assays validated by NMR structural data revealed that the MBSI sequence was bipartite. The first halfsite ((T/C)AAC)) has the majority of specific contacts with R3, and the second half-site ((G/T)G(A/C/T)(A/C/T)) had specific contacts with R2 [23,73,74]. Following identification of the c-MYB DNA-binding site, mammalian A-MYB and B-MYB, were subsequently shown to bind MBSI [14,75-77]. 3.2. Plant MYB DNA-binding sites Although R1R2R3–MYB proteins in plants share the same functionality as animal R1R2R3–MYB family members, their DNAbinding specificities are different [12,72,78]. All three characterised animal three-repeat MYB proteins bind to the same sequence MBSI ((T/C)AAC(G/T)G(A/C/T)(A/C/T)) and have similar functions in cellcycle control [10-12]. In comparison, plant three-repeat MYB proteins, such as tobacco MYBA1, MYBA2, and MYBB have an important role at the G2/M phase of the cell-cycle, by regulating transcription of cyclin B and other cell-cycle genes that are expressed at a similar time in the cell-cycle [79]. Through a yeast one-hybrid screen, NtMYBA1, NtMYBA2, and NtMYBB were found to bind to AACGG. This consensus sequence is known as the M phase-specific activator (MSA) element, and was identified previously in tobacco. The other two three-repeat MYB proteins found in tobacco, NtMYBC1 and

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NtMYBC2, are also predicted to bind to this motif; however, this assumption has not been validated experimentally. Relatively few of the possible plant R2R3–MYB DNA targets have been characterised; but some common elements of plant MYB–DNA interactions have emerged (Fig. 1, Supplemental Table S1). Recognition of plant MYB DNA targets was first determined with studies conducted on the Maize P protein, a R2R3–MYB protein involved in flavonoid biosynthesis [80]. Through binding-site selection assays and EMSAs, P was shown to bind to ACC(A/T)ACC(A/C/T). This contrasted with the animal MYB DNA consensus sequence of ((T/C)AAC (G/T)G(A/C/T)(A/C/T)), but was a harbinger for the majority of plant MYB proteins, which recognise MBSI ((T/C)AAC(G/T)G(A/C/T) (A/C/T)), MBSII (AGTTAGTTA), and MBSIIG ((C/T)ACC(A/T)A(A/C)C). Nevertheless, it is important to note that not all plant MYB proteins, especially within the R2R3–MYB family, recognise these motifs [81]. Many R2R3–MYB transcription factors recognise AC elements, DNA motifs that are enriched in adenosine and cytosine residues [37,62,63,80,82-90]. Some R2R3–MYB proteins function as transcriptional activators at these sites [62,63], while others function as transcriptional repressors [82]. Compendia of plant MYB DNA-binding sites can be found in databases such as The Arabidopsis Gene Regulatory Information Server (AGRIS) (http://arabidopsis.med.ohio-state.edu/) and the transcription factor database (TRANSFAC) (http://www.gene-regulation. com/pub/databases.html). These databases contain many of the MYB DNA-binding sites reported in the literature, most of which have been experimentally validated, and all of which are reported here (Fig. 1, Supplemental Table S1). Plant MYB DNA-binding sites were determined on a protein-by-protein basis [26,31,33,40,45,62,63,80,81,83,85,86,88-107], and generally reside approximately 500 bp upstream of the transcriptional start site (Fig. 1, Supplemental Table S1).

3.3. The DNA targets of single MYB-repeat proteins In contrast to two and three-repeat MYB proteins, single-repeat MYB proteins bind predominantly to the telomeric sequence TTAGGG and display similar sequence identity; however, not all single-repeat MYB proteins bind this sequence and moreover, they do not share the same functional roles (Supplemental Table S1). Single-repeat MYB proteins are involved with telomere binding and circadian clock regulation [15]. These functionalities have been conserved during the evolution of yeast, animals and plants [8,108]. Both C-terminal and N-terminal single-MYB repeat proteins bind to double-stranded DNA of telomeric repeats TTTAGGG. AtTRB1, AtTRB2, AtTRB3 all bind the telomeric DNA sequence containing a minimum of two repeats (TTTAGGG)2. In this regard, these single-MYB repeat proteins are divergent from R2R3–MYB and R1R2R3–MYB proteins both in terms of the primary sequence of the MYB domains, and also, consistent with their divergent DNA-binding domain, in terms of their cognate DNA target binding sites. By contrast, some single-repeat MYB proteins seem to bind to DNA targets that are coincident with R2R3– MYB proteins [36,45,47]. In this regard, they function as competitors for the same DNA targets. The rice single-MYB repeat proteins OsMYBS1, OsMYBS2, and OsMYBS3 can form dimers to bind to the sequence TATCCA with different binding affinities, as determined by EMSAs with competition [47]. Mutational assays showed that nucleotides CCA are more important for OsMYBS1 and OsMYBS3 binding than the TAT nucleotides. In contrast, sequence TAT seems to be more important for OsMYBS2 binding. Moreover, all three of these MYB proteins alter alpha-amylase gene expression. OsMYBS1 had a higher transactivation ability than OsMYBS2 and OsMYBS3. OsMYBS3 acted as a transcriptional repressor in both yeast and barley aleurone cells. These results demonstrate differential binding affinities and transactivation ability of three MYB proteins for the same target DNA sequence, showing the mechanism behind the phenotype. Competing with each other, single-repeat MYB proteins

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and R2R3–MYB proteins provide a means by which to fine-tune gene expression of genes that contain the gene regulatory regions that are the sites of competition [42,45,47]. 4. The nature of DNA-binding by MYB proteins 4.1. Relationship between the MYB DNA-binding domain and DNA-binding specificity The MYB superfamily has been categorised based both on the number of MYB repeats and on the amino acid sequence of the MYB domain [21,24]. In other families of transcription factors, overall sequence conservation is low and variability in DNA-binding specificity is high [109,110]. Contrary to this, members of the plant R2R3–MYB family share higher amino acid sequence similarity, especially in their recognition helices, and display similar DNA-recognition patterns [81]. These similarities in recognition specificity are heightened between members of the same phylogenetic group. R2R3–MYB family members from different species have been previously classified into different phylogenetic clades (groups A, B, and C) based on sequence similarities [81]. These clades were then analysed for DNA-binding specificities [81]. The R2R3–MYB DNA-binding data was mainly a result of in vitro assays; however, in vivo assays were used as well to determine R2R3–MYB DNA-binding specificities. It was shown that members from group A, bind MBS type I sequence (C(A/C/G/T)GTT(A/G)), members of group B bind equally to both MBS type I and type II (G(G/T)T(A/T)GTT(A/G)), and most members of group C bind MBS type IIG ((C/T)ACC(A/T)A(A/C)C). For example, AtMYB6 and AtMYB7 are both members of group C and share 90% amino acid sequence identity [81]. AtMYB6 and AtMYB7 both bind to the MBS type IIG sequence [97] (Fig. 1, Supplemental Table S1). Well-characterised DNA-binding sites can be extracted from the literature for 87 proteins from the MYB superfamily (Fig. 1, Supplemental Table S1). Characterisation of DNA targets was derived from both in vivo and in vitro protein–DNA-binding assays (see captions for Fig. 1 and Supplemental Table S1). DNA binding sites for these proteins can be categorised into seven groups, and each of the 87 MYB proteins can be grouped based on DNA-binding specificities. Examinaton of the protein-sequence-similarity-derived phylogenetic relationships between these 87 MYB proteins reveals that, in general, MYB proteins that share protein sequences bind to similar DNA sequences (Fig. 1, Supplemental Table S1). That is, similar protein structure implies similar DNAbinding sequences recognized by MYB proteins; however, there are instances in the phylogenetic tree and in other studies where this is not the case [97] (Fig. 1, Supplemental Table S1). Members of the MYB superfamily do not always share similar DNAbinding sites based on similar structure. Although Romero et al. had shown a correlation between MYB protein structure and DNA-binding specificity, there were MYB family members that did not fit into his predictions [81]. For example, Group C MYB family members prefer in general type IIG sequence; however, the two Group C MYB proteins, AtMYB2 and AtMYBGL1, bound DNA with different patterns. AtMYB2 bound to type I sequences [105], and AtMYBGL1 bound only to type II sequence [81]. These examples show that MYB proteins, although similar in structure and function, can bind to different DNA cognate target sites. Moreover, it highlights the importance of conducting DNAbinding site experiments for individual MYB proteins because it is extremely difficult to predict a MYB DNA-binding site based solely on homology. 4.2. Involvement of MYB repeats in DNA binding Both R2 and R3-MYB repeats are necessary for DNA binding, either by R1R2R3–MYB or R2R3–MYB proteins [23]. Neither R2 nor R3 can alone bind DNA specifically [23]. This implies that both the R2 and R3 repeats bind cooperatively to its cognate DNA target sequence.

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Fig. 1. Phylogenetic relationships and subgroup designations for 87 MYB superfamily members using the neighbour-joining method. The unrooted phylogenetic tree was generated using the amino acid sequences of the MYB proteins in Supplemental Table S1. Whole MYB protein sequences were downloaded from The Arabidopsis Information Resource (TAIR; http://Arabidopsis.org) and from the National Center for Biotechnology Information protein database (NCBI Entrez; http://www.ncvi.nlm.nih.gov/sites/entrez). The phylogenetic analysis included 9 three-repeat MYB proteins (R1R2R3–MYB proteins), 50 two-repeat MYB proteins (R2R3–MYB proteins) and 28 one-repeat MYB proteins (R1-MYB proteins). The full-length amino acid sequences were aligned using Multiple Alignment using Fast Fourier Transform (MAFFT) using the G-INS-I algorithm [179]. A neighbour-joining tree was constructed using Molecular Evolutionary Genetics Analysis 4 (MEGA 4) [180] with the parameters for the Jones-Taylor-Thornton substitution model and a Gamma parameter of 1.0 to account for the uneven rates of substitution across the length of the MYB proteins. Pairwise gap deletion was used, along with a bootstrap value of 1000. DNA-binding sites for MYB proteins were obtained from the literature. MYB proteins are annotated by colour based on DNA sequence recognition. Red, blue, green, orange, purple and grey represent MYB proteins that bind CNGTT(A/G), ACC(A/T)A(A/C), TTAGGG, AAAATATCT, GATA and TATCCA respectively. Black represents MYB proteins that do not bind to an assigned group. N indicates adenine, guanine, cytosine or thymine. * indicates that the MYB protein DNA-binding specificity differs slightly from the consensus sequence of its group. Refer to Supplemental Table S1 for specific details on DNA sequences bound by the MYB proteins.

The resolved structure of the R2R3–DNA complex has displayed that both R2 and R3 recognition helices contact directly with each other prior to sequence-specific binding [23,111-113]. Furthermore, the phosphate backbone interacts simultaneously with the amino acids in both the R2 and R3 repeats to aid in DNA-binding. As R1 is not necessary for the specific recognition of DNA target sequences, both R1R2R3 and R2R3–MYB proteins bind DNA in a similar manner. By contrast, single-repeat MYB proteins, which only possess one MYB DNA-binding repeat, bind DNA in a different manner than R1R2R3 and R2R3–MYB proteins [38]. This first became clear when S. cerevisiae Rap1 was found to contain two MYB repeats in its MYB–DNAbinding domain and its orthologous MYB counterpart, Homo sapien RAP1, only possessed one MYB repeat [42]. It was subsequently found that S. cerevisiae Rap1 binds DNA as a monomer because it contains two MYB repeats. In contrast, H. sapien RAP1 contains only one MYB repeat and does not bind DNA directly; however, it tethers itself to shelterin via TRF2 to bind DNA in a sequence specific manner.

4.3. The nature of DNA binding by animal MYB proteins The nature of DNA binding by any MYB protein has been most extensively examined using c-MYB and its cognate target, MBSI. Mutational studies on c-MYB have shown that R1 can be deleted without significant loss of DNA-binding ability, and that both R2 and R3 are essential for MYB–DNA recognition and binding [114-116]. Although R1 is not involved in the direct recognition of DNA sequence motifs, it does enhance the stability of DNA binding by the R2R3 repeats without significantly altering the DNA–R2R3 conformation. The R1 repeat is hypothesised to achieve this by fluctuating between bound and unbound states at a rate faster than the chemical shift time scale [23,74]. c-MYB–DNA interactions were validated structurally with the resolution of the NMR solution structures and X-ray crystal structures of c-MYB DNA-binding domain in the free and DNA-bound states [23,111-113]. Each third helix (C-terminal helix) of R2 and R3 were subsequently found to act as the recognition helix [23]. In keeping

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with this, the recognition helix of R3 interacts with the core of the DNA consensus sequence ((T/C)AAC)); while the recognition helix of R2 interacts less specifically with nucleotides surrounding the core recognition motif ((G/T)G(A/C/T)(A/C/T)) [111]. The binding of R2 and R3 to its consensus sequence ((T/C)AAC(G/T)G(A/C/T)(A/C/T)) widens the major groove and causes a bend of local helical axis [23]. Several interhelical interactions occur between the helices of R2 and R3, stabilising the MYB–DNA interaction. Moreover, R2 and R3 bound the major groove continuously, similar to transcription factor IIIA (TFIIIA)-type Zn fingers [117-119]. Contrary to TFIIIA-type Zn fingers, the recognition helices of c-MYB R2 and R3 are more closely packed together in the major groove. This type of direct interaction between the recognition helices from different DNA-binding units is unique among other DNA-binding domain complexes [23]. Not all amino acid residues within the DNA-binding site of transcription factors partake in DNA recognition and binding. Within the MYB protein family, certain key residues are critical for these tasks [23,103]. For example, for c-MYB, the three key base contacts are governed by residues Lys128 (R2), Lys182 (R3), and Asn183 (R3), which are found to be fully conserved in all know animal and plant MYB proteins [23,111]. Each MYB DNA-binding domain contains several conserved regularly spaced tryptophan residues that participate in a hydrophobic cluster [114,120]. Through mutational and structural studies on cMYB, this hydrophobic cluster was determined to be essential for both the stability of MYB-protein interaction and for sequencespecific binding to its consensus sequence ((T/C)AAC(G/T)G(A/C/T) (A/C/T)). Mutational and structural studies on animal c-MYB have aided in providing critical knowledge on the molecular mechanisms behind MYB–DNA interactions. Moreover, these studies allow one to generate testable hypotheses on MYB–DNA interactions in other organisms where orthologous MYB proteins reside. A cysteine residue located in the DNA recognition helix of R2 has remained completely conserved in animals, fungi, and plants during the evolution of MYB domains [121]. R1R2R3–MYB domains have a cysteine residue (Cys130) that is included in the hydrophobic core. Cys130 of c-MYB needs to be reduced to allow for sequence-specific DNA-binding. When reduced, Cys130 accomplishes this by structurally stabilising the three helices of the R2-MYB repeat during sequence-specific DNA-binding [122-124]. In contrast, most R2R3– MYB domains possess two cysteine residues (Cys49 and Cys53) with the equivalent position as Cys130 in R1R2R3 MYB [121]. c-MYB has been extensively studied with regards to dynamics of DNA binding [23,74,111,125]. The c-MYB R2R3-domain was shown to bind tightly to the MYB binding site ((T/C)AAC(G/T)G(A/C/T)(A/C/T)) with a binding constant of 1.5E− 09 M ± 28% [74,125]. Mutational analyses have shown that specific residues within the R2R3–MYB repeats of c-MYB bound to specific nucleotides with different affinities [74]. Specific interactions within the R2R3–MYB repeats of c-MYB are disproportionately localised in the AACTGAC region of the MYBbinding site. The first adenine, the third cytosine, and the fifth guanine are involved in extremely specific interactions with c-MYB, in which any base substitutions reduce the binding affinity by more than 500fold. In contrast to this, the interaction with the second adenine is less specific, with an affinity reduction in the range of 6 to 15-fold when subjected to base change. The seventh cytosine shows an interesting interaction in that only guanine substitution abolishes the specific binding. All together, these affinity data show that the second and third MYBrepeats cover the AACTGAC region from the major groove of DNA in an orientation that allows the third MYB-repeat to cover the core AAC sequence. Moreover, the results show that the third MYB-repeat recognises the core AAC sequence very specifically; however, the second repeat recognises the GAC sequence in a redundant manner. MYB–DNA kinetic studies also found that mutating the R1 repeat does not affect the DNA recognition of c-MYB but does effect the stability of the MYB–DNA complex. Furthermore, the N-terminal

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acidic activation region upstream of the first MYB repeat was found to reduce the binding affinity by interfering with R1 binding to DNA. NMR, X-ray crystallography, and surface plasmon resonance studies have validated these c-MYB–DNA binding kinetic results [23,111,126]. Further studies on c-MYB DNA affinity indicated that when c-MYB binds DNA, the orientation of R2 and R3 are immobilised by sequence-specific binding and their conformations are slightly changed. No significant conformational changes occur in R1 during MYB DNA-binding, further emphasising that R1 is not involved in DNA-binding site recognition [111]. In a comparison between the binding kinetics of the three vertebrate MYB proteins (A-, B- and c-MYB), both A- and c-MYB bound the MYB recognition site with similar binding constants and specificity; however, B-MYB formed DNA-protein complexes of lower stability, rapidly dissociating under competitive conditions and showed less tolerance to DNA-binding site variations [127]. These studies on animal MYB proteins have granted insight into the molecular mechanisms behind MYB–DNA interactions in general because R2R3–MYB proteins bind DNA in a similar fashion [23,103]. Kinetics on single-repeat animal MYB proteins binding to their DNA cognate sequences have also been examined. For example, the human single-repeat MYB protein TRF1 shows that TRF1 can bind to the telomeric sequence TTAGGG with high affinity (Kd = 3.2 ± 0.5 × 10 − 9 M) and specificity as a monomer [7]. The recorded DNA binding affinity lies in the range of various homeodomains that also bind specifically to DNA as monomers [128-131]. Although the interaction of TRF1 and the telomeric sequence is specific, the specificity and affinity is significantly increased as a homodimer [132]. 4.4. The nature of DNA binding by plant MYB proteins To date, some of the specifics of plant MYB interaction with target DNA have relied on model-building based on the c-MYB binding to DNA, as no crystal structure has been generated yet for any plant multi-repeat MYB protein. For example, PAP1/AtMYB75, the R2R3domain was modelled according to the known structural data of c-MYB [133]. A conserved amino acid signature ([DE]Lx2[RK] x3Lx6Lx3R) found within several MYB proteins was hypothesised to predict new MYB/BHLH interactions for A. thaliana proteins. Consistent with this hypothesis, analysis of the predicted 3D model of PAP1/AtMYB75 showed that the amino acids of the conserved motif are surface-exposed on helices 1 and 2 of the R3 repeat, forming hydrophobic and charged residue patterns [133]. These surface-exposed amino acids are thought to stabilise the protein–protein interactions [133]. This model was validated by mutational assays [133]. The Petunia MYB Ph3 structure was also modelled after c-MYB. MYB. Ph3, a plant R2R3–MYB transcription factor involved in the regulation of flavonoid biosynthetic pathway in petunia flowers [85,134,135], shows divergence in binding specificity compared to c-MYB [103]. MYB.Ph3 can bind two types of sites: MBSI ((T/C)AAC(G/T)G(A/C/T)(A/C/T)) and MBSII (AGTTAGTTA) [81,103]. Modelling predicted that a single residue substitution in the R2 repeat of MYB.Ph3 (Leu71►Glu) would change its DNA recognition to that of c-MYB, and the reciprocal substitution in c-MYB, Glu132►Leu would change c-MYB specificity to that of MYB.Ph3 [103]. This model was experimentally validated via mutational assays. Even though it was previously found that these residues do not directly bind DNA [23], the MYB.Ph3 Leu71 and c-MYB Glu132 residues interact with residues that do interact with DNA, enabling them to impact DNA-specificity indirectly [103]. By contrast, Williams and Grotewold [136] found that P and v-MYB DNAbinding domains, which are conserved among animal and plant MYB domains, are necessary for the high affinity DNA-binding activity of these proteins to their respective DNA target sites but are not sufficient for their unique DNA-binding site recognition of P and vMYB. Furthermore, Williams and Grotewold [136] found that chimeric MYB domains have novel DNA-binding specificities. Resolution of

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these differences will require crystal or solution structures for plant MYB proteins. As is the case with c-MYB, both Cys49 and Cys53 are thought to be essential for the DNA-binding or transcriptional activity of plant MYB proteins, forming an intramolecular disulfide bond with each other under non-reducing conditions. This disulfide bond has been hypothesised to impair DNA binding under non-reducing conditions, causing R2R3–MYB proteins to be functionally active only under reducing conditions. Toward this end, the same two cysteines are conserved in the R2-MYB repeat of the R2R3–MYB protein WEREWOLF (WER) [95]. WER cannot bind to its DNA-binding sites within its downstream target promoter regions without the addition of dithiothreitol (a reducing agent). The dithiothreitol is thought to abolish the disulfide bond, leading to the sequence specific binding of WER to its downstream targets. Nitric oxide (NO) was shown to modifiy the DNA-binding activity of AtMYB2 by a posttranslational modification of its conserved Cys53 [137]. AtMYB2 bound to the core binding site AAACCA in an EMSA assay; however, the addition of NO donors, such as SNP (sodium nitroprusside) and GSNO (S-nitrosoglutathione), inhibited sequence specific binding of AtMYB2. The NO-mediated inhibitory effect was reversed by DTT, demonstrating that sequence specific DNA-binding of AtMYB2 is inhibited by S-nitrosylation of Cys53 as a result of NO action. The role of cysteine residues in MYB proteins displays the divergence of DNA binding mechanisms between both animal and plant MYB proteins. Despite some similarities in DNA binding, given the divergence of target DNA-binding sites of R2R3–MYB proteins relative to R1R2R3–MYB proteins, it follows that the residues critical for DNA recognition and binding within the binding site of many plant MYB proteins differ from those of animal MYB proteins. These examples display why there is inherent flexibility of DNA recognition by the MYB superfamily of transcription factors because merely one change in residue can alter the DNA recognition by a particular MYB protein. Despite the vast knowledge of plant MYB transcription factor function at the gross morphological level, little is known about the dynamics of MYB protein–DNA interactions. Nevertheless, some general themes regarding plant MYB–DNA binding kinetics are emerging [45,47,103]. Most plant MYB proteins display considerable inherent flexibility in their ability to recognise target sites (Fig. 1, Supplemental Table S1). For example, Petunia protein MYB.Ph3 bound to both MBSI and MBSII sites with the same affinity, inducing similar DNA-bending/ distortions in both cases [103]. Affinities for these two plant MYB binding sites vary among other plant MYB proteins; however, certain MYB proteins have been shown to only bind one of these sequences [80,83,85,92,97,105]. The maize R2R3–MYB C1 protein bound to its target sequences in the a1 (dihydroflavonol reductase) promoter [86]. Determined by EMSA assays, the affinity of binding was reduced by mutations in the C1 DNA-binding domain or in the a1 sequences recognised and bound by C1. Maize transient assays determined that C1 directly activated the a1 gene. Towards this end, the two C1 binding sites were also bound by the maize P protein. One of the sites (ACC(A/T)ACC) were bound with higher affinity by P (Kd = 52 ± 4 × 10− 9 M) relative to C1 (Kd = 330 ± 50 × 10− 9 M). In contrast, the other site (AACTACCGG) is bound with similar low affinities by P (Kd = 860 ±150 × 10− 9 M) and C1 (Kd = 780 ± 70 × 10− 9 M). These results allow a greater understanding of the mechanism behind the anthocyanin biosynthetic pathway in maize. In another example, all three Soy-MYB proteins, GmMYB76, GmMYB92, and GmMYB177, bound to the MBSI sequence [45]. GmMYB92 could also bind sequences MRE4 (TCTCACCTACC) and mMRE1 (CCGGAAAAAAGGAT). Unlike GmMYB92, GmMYB76 and GmMYB177 bound to the mMRE1 sequence with weak affinity. It is important to note that, while the aforementioned studies have provided profoundly useful insights into plant R2R3–MYB interactions with DNA sequences, they also provide a rather incomplete picture of the specific interactions that are possible. Given the sheer number of plant MYB proteins, the correspondingly large number of downstream

DNA targets for these proteins, and the breadth of processes controlled by the MYB family members in plants, the complexity of plant MYB–DNA interactions characterised to date is the tip of the proverbial iceberg. Clearly, there is a need for more extensive analysis of these important interactions. One might expect considerable inroads to be made in future, with the emergence of new technologies to probe DNA-protein interactions. 5. Future of plant MYB–DNA interaction studies 5.1. Determining the breadth of MYB DNA targets in vitro The identification of in vitro MYB DNA-binding sequences in a rapid and high-throughput manner is required in the future to identify all variants of their DNA targets. Transcription factors, including MYB proteins, are promiscuous in terms that they can interact and initiate transcription from multiple target sequences [63,103,138]. Well-established protocols based on recombinant MYB transcription factor (TF) DNA-binding domains have been used to enrich for target sequences from libraries of random DNA sequences [12,72,80,139]. These experiments include cyclic amplification and selection of targets (CASTing) [140] and systematic evolution of ligands by exponential enrichment (SELEX) [141]. Both of these procedures have determined numerous MYB in vitro DNA binding motifs for several MYB transcription factors, and their underlying principles can now be scaled to accommodate high-throughput approaches. Microarray based technologies, such as protein-binding microarrays, have been developed to identify transcription factor sequence specificities [142-145]. Binding sites identified by this technology have correlated with in vivo transcription factorbound DNA sequences identified by ChIP experiments [144,146,147]. Two types of protein-binding microarrays have emerged: doublestranded DNA microarrays and transcription factor microarrays. Double-stranded DNA microarrays possess all possible doublestranded 11 bp sequences (approximately 4.2 million sequences) in roughly 240,000 oligonucleotides [148]. Recombinant protein from a transcription factor of interest is flowed over the double-stranded DNA microarray and washed with increasing concentrations of salts. This technology allows the accurate quantification of binding affinities to all possible DNA-binding sites recognised by the transcription factor of interest in just one hybridization step. Transcription factor DNA-binding enrichment, based on a protein array, allows for the capture multiple transcription factors and then discovery of their binding sites [149]. A library of random oligonucleotides is flowed over captured proteins to identify the transcription factors’ DNA-binding sites. The array is then washed with increasing salt concentrations to allow for the identification of relative binding affinities. This specific protein-binding microarray has a slight advantage over the double-stranded DNA microarray because multiple transcription factors can hybridise onto a chip, allowing for the identification of binding preferences for transcription factor families [150]. Both these techniques are powerful means to identify in vitro DNA-binding sites of proteins of interest in a time efficient manner [149,150]; however, there are limitations to these experiments. One limitation is that protein–DNA complexes that have weak affinity for each other will be washed away with low concentrations of salts, biassing the results. Another limitation is that the whole structure of the protein is not available to bind to its preferred DNA targets because a portion of the protein is hybridised to the array. Not knowing the DNA binding domain of the protein of interest could lead to misleading results. 5.2. Emerging approaches for plant MYB target discovery and analysis in vivo Crucially, MYB DNA-binding sites in vitro might differ from those preferred in vivo [151,152]. These differences are a result of in vivo protein–protein interactions and post-translational modifications

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altering DNA binding specificity, as well as conformational differences between in vitro recombinant DNA-binding domain and in vivo native conformations of these domains. The in vivo availability of transcription factor DNA-binding sites is also controlled by the packaging of genomic DNA in chromatin. Therefore, alternative in vivo approaches are necessary to map MYB–DNA binding sites in the genome accurately. Transient expression assays and yeast one-hybrid assays are now a staple in identifying that a particular MYB binds to a specific DNA target in vivo [62,63,153]. These procedures involve the expression of a transcription factor of interest within organisms, such as plants or yeasts, to see if it is sufficient to enable the transactivation of an artificial gene comprising a tandem repeat of its putative DNAbinding site fused to a minimal promoter, upstream of a reporter gene. These experiments, with the right controls, ensure that a specific transcription factor of interest interacts and activates transcription from its putative DNA binding site in vivo. For example, the R2R3–MYB transcription factors AtMYB11, AtMYB12 and AtMYB111 were shown, through transient expression assays in Arabidopsis protoplasts, that they were functionally similar to its structurally similar maize P protein [154,155]. AtMYB11, AtMYB12, AtMYB111, and P protein had similar target gene specificity, regulating a myriad of flavonoid biosynthetic genes. Furthermore, all activated target gene promoters in vivo in the presence of a MYB recognition element. Transient expression assays and yeast one-hybrid assays are well established experiments to validate if a particular protein activates transcription from a particular motif; however, chromatin immunoprecipitation (ChIP) followed by either whole-genome tiled microarray analysis (ChIP-chip) or high-throughput signature sequencing (ChIP-seq) can identify novel in vivo DNA targets of proteins of interest, resulting in more biologically significant results. ChIP-chip or ChIP-seq has proven to be powerful tools by which to identify in vivo binding sites of sequence-specific transcription factors in the context of chromatin [156], which avoids many caveats of the aforementioned techniques [157]. Recently, ChIP identified in vivo DNA-binding target sites for a select group of MYB proteins [153,158-163]. In plants, ChIP-chip identified in vivo binding sites for two Arabidopsis two-MYB-repeat proteins, FOUR LIPS (FLP; AtMYB124) and AtMYB88 [153]. FLP and MYB88 were shown to directly bind promoters of cell cycle genes, including CDKA:1 (At3g48750), CELL DIVISION CYCLE6a and 6b (CDC6a At2g29680 and 6b At1g07270), Cyclind4:1 (At5g65420), a cyclin-like gene, CYCLINT:1 (CYCT:1, At1g35440), CDKD1:3 (At1g8040) and CYCB1:3 (At3g11520). These results were consistent with FLP/ MYB88 in suppressing DNA replication and cell cycle progression within the stomata. Systematic evolution of ligands by exponential enrichment (SELEX) and EMSA, along with ChIP-chip, helped identified that this group bound to the core consensus sequence of (A/T/G)(A/T/ G)C(C/G)(C/G). Similarily, Zea mays MYB31 was shown by SELEX and ChIP to bind to the sequence ACC(T/A)ACC within the two lignin promoters XmCOMT and ZmF5H, resulting in the repression of lignin biosynthetic gene expression [159]. Furthermore, ChIP-chip was performed on the trichome developmental selectors GLABRA3 (GL3) and GLABRA1 (GL1), encoding basic helix-loop-helix (bHLH) and MYB transcription factors respectively. ChIP-chip identified 20 novel in vivo GL3/GL1 direct targets such as SCL8 and MYC1 (involved in the control of gene expression), SIM (a cyclin-dependent kinase inhibitor), and RBR1 (a negative regulator of the cell cycle transcription factor E2F) [162]. ChIP-chip and ChIP-seq are difficult procedures to conduct because these procedures require an antibody that specifically recognises a transcription factor of interest. Although these procedures are the most effective way in determining true in vivo DNA-binding targets and sites, they have not been used to study the majority of the MYB superfamily. Epitope tagging is the process of making the product of a gene of interest immunoreactive to an already synthesised antibody [156].

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This can be done by inserting a polynucleotide encoding an epitope into a gene of interest and expressing the gene in an appropriate host. This protein from the gene of interest can now be located via an antibody that has already been generated. This method could be used as an alternative to generating novel antibodies before conducting a ChIP-chip. When an antibody cannot be generated to a protein of interest, this method is best used to determine in vivo DNAprotein binding data on the protein of interest. Other methods can also identify in vivo MYB DNA-binding sites and downstream targets. A glucocorticoid receptor (GR)-mediated inducible system has successfully been used to define direct target genes of several putative transcription factors [164-166]. In a GR-mediated inducible system a fusion protein between a protein of interest and the rat glucocorticoid receptor hormone binding domain is engineered. This fusion protein is retained in the cytoplasm in absence of the synthetically made steroid hormone dexamethasone. Upon addition of dexamethasone the protein of interest-GR fusion protein enters the nucleus and binds to the protein of interest's downstream target genes. The addition of translational inhibitors such as cycloheximide will inhibit further downstream effects of your protein of interest. By assaying genome-wide expression changes on microarrays, one can determine direct target genes of a protein of interest. The GR inducible system was used to show the single-repeat MYB protein CAPRICE (CPC) transcription is regulated directly by WER (a R2R3– MYB transcription factor). Using EMSAs, two WER-binding sites (WBSs; WBSI and WBSII) were verified in the CPC promoter. WERWBSI binding was further validated in vivo using yeast one-hybrid assays. In another example, AtMYB80 involvement in tapetal and pollen development was examined [167]. Using the GR system, it was determined that 79 genes were changed when the R2R3–MYB transcription factor AtMYB80 function was restored in the myb80 mutant following dexamethasone induction [167]. Thirty-two of these genes were analysed using ChIP, and three were identified as direct targets of AtMYB80. These genes were shown to encode a glyoxal oxidase (GLOX1), a pectin methylesterase (VANGUARD1), and an A1 aspartic protease (UNDEAD) and corresponded with in vitro binding data. This procedure is a powerful way of identifying direct targets of transcription factors. When the GR-inducible system is coupled with in silico processes, such as promoter analyses, and DNA-binding experiments, such as EMSAs, yeast-one hybrid assays and protoplasts assays, it has proven quite useful in identifying in vivo DNA binding sites. The use of both expression data and DNA-binding site experiments are required to reduce the amount of MYB false positive targets. The forkheadboxA homolog, PHA-4 regulates organogenesis of the pharynx in Caenorhabditis elegans [168]. Expression of PHA-4 targets correlated with its binding sites in promoter regions, and that the timing of target expression correlated with binding affinity between PHA-4 and its target sequence [169]. The data suggested that PHA-4 regulates pharyngeal organ development by combining PHA-4 binding affinity and cooperating factors to regulate gene expression temporally. ChIP-seq data for PHA-4 validated this assessment — 87% of the associated genes were expressed when PHA-4 binding was present, and this expression was reduced to 60% when PHA-4 binding was not present [170]. Towards this end, using both expression data and DNA-binding site experiments is a powerful means of validating that the binding of a factor activates the expression of its putative target genes. The use of both expression data and MYB–DNA binding site assays in tandem will aid in generating a more biologically significant MYB network. Another problem with the identification of MYB binding sites is that some MYB binding sites are not proximal to the predicted target genes. In an early ChIP-seq study, there were a large number of bound sites observed for the human interferon-g (IFN-g) responsive transcription factor STAT1 [171]. Before stimulating the cells with IFN-g, 10,000 binding sites were identified. Binding sites for STAT1

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increased fourfold after stimulating the cells with IFN-g. In both conditions, approximately 50% of the total sites were intragenic, 25% of the total sites were intergenic. Most binding sites were not located near STAT1-regulated genes, which suggested that bound sites were not directly regulating nearby genes. This can be explained by chromatin looping - a mechanism for transcriptional control that involves bringing regulatory elements into proximity of target genes [172]. The use of chromosome conformation capture studies in the future will determine if the distant locus control region (LCR) with MYB target genes are required for high-level transcription. 6. Conclusion The past decades have seen remarkable inroads made into our understanding of the molecular interactions between sequence-specific transcription factors and their DNA targets in general, and MYB proteins and their binding sites more specifically. This is particularly true for animal R1R2R3–MYB transcription factors. Insights into the specificities of R1R2R3–MYB interactions with DNA target sequences is, in turn, providing greater understanding of the molecular mechanisms that control cellular processes [12,62,72,80,103] (Fig. 1, Supplemental Table S1), and is aiding in the development of diagnostics and therapeutics for when those mechanisms go awry [173,174]. By contrast, comparable understanding of the molecular mechanisms that proceed from the interaction of plant R2R3–MYB proteins with their cognate DNA targets is much less complete, with only a handful of MYB–DNA interactions characterised at the molecular level for any given plant species. Consequently, the precise means by which plant R2R3–MYB proteins coordinate gene expression to give rise to plant phenotype is still at a relatively nascent stage. This said, with the emergence of new techniques that enable the dissection of protein– DNA interactions more rapidly [175,176], and/or with higher resolution [156,177], and/or for a larger number of proteins [145,148,178], the characterisation of new MYB–DNA interactions, particularly those that occur in plant species, should proceed apace. Given this, the coming decade promises to provide great insights into the means by which members of this remarkable family of proteins convert molecular information into whole organism responses. Supplementary materials related to this article can be found online at doi:10.1016/j.bbagrm.2011.10.010. Acknowledgements We are very grateful to Dr. Katharina Bräutigam, Joseph Skaf, and Heather Wheeler for fruitful discussions and extensive assistance with earlier drafts of this manuscript. This work was generously supported by a Natural Science and Engineering Research Council of Canada (NSERC) Canadian Graduate Scholarship (CGSD) awarded to MP, and by funding from the University of Toronto and NSERC to MMC. References [1] T. Maniatis, S. Goodbourn, J.A. Fischer, Regulation of inducible and tissuespecific gene expression, Science 236 (1987) 1237–1245. [2] J. Colladovides, B. Magasanik, J.D. Gralla, Control site location and transcriptional regulation in Escherichia coli, Microbiol. Rev. 55 (1991) 371–394. [3] C.O. Pabo, R.T. Sauer, Transcription factors: structural families and principles of DNA recognition, Annu. Rev. Biochem. 61 (1992) 1053–1095. [4] C. Yanhui, Y. Xiaoyuan, H. Kun, L. Meihua, L. Jigang, G. Zhaofeng, L. Zhiqiang, Z. Yunfei, W. Xiaoxiao, Q. Xiaoming, S. Yunping, Z. Li, D. Xiaohui, L. Jingchu, D. Xing-Wang, C. Zhangliang, G. Hongya, Q. Li-Jia, The MYB transcription factor superfamily of Arabidopsis: expression analysis and phylogenetic comparison with the rice MYB family, Plant Mol. Biol. 60 (2006) 107–124. [5] J.L. Riechmann, J. Heard, G. Martin, L. Reuber, C.Z. Jiang, J. Keddie, L. Adam, O. Pineda, O.J. Ratcliffe, R.R. Samaha, R. Creelman, M. Pilgrim, P. Broun, J.Z. Zhang, D. Ghandehari, B.K. Sherman, C.L. Yu, Arabidopsis transcription factors: genome-wide comparative analysis among eukaryotes, Science 290 (2000) 2105–2110.

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