Differences between Plant and Animal Myb Domains Are Fundamental for DNA Binding Activity, and Chimeric Myb Domains Have Novel DNA Binding Specificities*

(Received for publication, September 9, 1996, and in revised form, October 23, 1996)

Christopher E. Williams Dagger and Erich Grotewold §

From Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724-2212 and Dagger  Dowling College, Oakdale, New York 11769-1999

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
Acknowledgments
REFERENCES


ABSTRACT

Several Myb domain proteins have been identified in plants, in which they play important regulatory roles in specific cellular processes. Plant and animal Myb domains have significant differences, but how these differences are important for function is not yet understood. The P gene encodes a Myb domain protein that activates a subset of flavonoid biosynthetic genes in maize floral organs. P and v-Myb bind different DNA sequences in vitro. Here we show that the Myb domain is solely responsible for the sequence-specific DNA binding activity of P, which binds DNA only in the reduced state. Differences in the DNA binding domains of v-Myb and P, which are conserved among animal and plant Myb domains, are fundamental for the high affinity DNA binding activity of these proteins to the corresponding binding sites but are not sufficient for the distinct DNA binding specificities of P and v-Myb. We conclude that significant structural differences distinguish plant from animal Myb domains. A chimeric Myb domain with a novel DNA binding specificity was created by combining Myb repeats of P and v-Myb. This approach could be used to artificially create novel Myb domains and to target transcription factors to genes containing specific promoters or to modify Myb-mediated interactions with other cellular factors.


INTRODUCTION

Transcription factors are classified in structural families according to the presence of specific DNA recognition motifs (1). Whereas basic structural characteristics among members of a family are conserved in all organisms, little is known about how functional constraints limit the evolutionary variability within a given family. Proteins containing the Myb DNA binding domain have been identified in organisms as diverse as fungi (2, 3, 4), Xenopus (5), Drosophila (6), plants (7, 8, 9, 10, 11, 12, 13, 14), chickens (15), mice (16), and humans (17, 18). Mouse and human forms of c-Myb have been proposed to play fundamental roles in cell cycle control and in the proliferation and differentiation of hemopoietic cells. Several lines of evidence suggest a role for Myb as a transcriptional regulator (reviewed in Ref. 19). To date, the only gene identified as a direct target of c-Myb is Mim-1 (20), and the lack of other known Myb-regulated genes has hindered the study of the mechanisms by which Myb carries out its cellular functions.

In plants, a large number of Myb domain proteins have been identified and shown to participate in a variety of important cellular functions (11, 14, 21, 22). Petunia contains a Myb gene family with at least 40 members (12). In Arabidopsis thaliana, more than 100 Myb-homologous sequences have been identified (23), and at least 20 different Myb domain genes are expressed in Arabidopsis flowers.1 The maize Myb domain proteins P and C1 regulate the accumulation of related, but different, flavonoid-derived pigments (24) through the activation of overlapping sets of flavonoid biosynthetic genes (25, 26). Maize flavonoid biosynthesis is probably one of the best described plant metabolic pathways, and studies of several putative target genes for the P and C1 proteins have yielded important information (27). In particular, P activates transcription of A1, one of the genes regulated by both P and C1, by binding to the P-responsive elements identified in the A1 gene promoter (26). Studies have also shown that the DNA consensus sequence CCT/AACC recognized by P (26) differs substantially from the sequence C/TAACGG recognized by animal Myb proteins (26, 28, 29).

Myb domains are usually formed by two or three imperfect 51- or 52-residue repeats (R1, R2, and R3).2 Each repeat encodes three alpha  helices, with the second and third helices forming a helix-turn-helix (HTH) structure when bound to DNA, similar to motifs found in the lambda  repressor and homeodomain proteins (30, 31, 32, 33). R2 and R3 are sufficient for sequence-specific DNA binding (31, 34, 35), and although the c-myb proto-oncogene contains all three repeats, retroviral versions of c-Myb (v-Myb) contain only R2 and R3 (36), as do most plant Myb domain proteins (12).

The structure of the mouse c-Myb R2-R3 DNA binding domain bound to DNA has been determined by NMR. The two Myb repeats R2 and R3 are closely packed in the major groove, with the third helix of each repeat making contact with DNA such that the recognition helices contact each other to bind to DNA in a cooperative manner (33). P and several other plant Myb domain proteins differ from v-Myb and many other animal Myb domain proteins in several aspects, which include the presence of an additional leucine residue between the second and third helices of R2 and residue differences at conserved positions in each of the two DNA recognition helices. Whereas the NMR studies of the c-Myb R2-R3 DNA binding domain offered important insight into Myb domain structure, they did not address the significance of the conserved differences between Myb domains of different kingdoms in Myb domain structure or sequence-specific DNA recognition.

Here we show that the highly conserved Myb domains of P and v-Myb are sufficient for conferring differential DNA binding properties to these proteins in vitro. P requires both of its Myb repeats and must be in the reduced state (REDOX) to bind DNA efficiently. Differences in the DNA recognition helices of P and v-Myb, conserved among plant and animal Myb domains, respectively, play fundamental roles in permitting these proteins to bind to their corresponding target sites in vitro, providing the first experimental evidence that plant and animal Myb domains are structurally different. A chimeric Myb domain with a novel DNA binding specificity was created by combining Myb repeats of P and v-Myb in a specific orientation. This illustrates the possibility of artificially designing and creating novel Myb domains, useful for targeted gene regulation by binding to specific promoter elements or by Myb-mediated interactions with other cellular factors, providing convenient tools for both basic and applied studies.


EXPERIMENTAL PROCEDURES

DNA Manipulations

Standard cloning techniques were used (37). Mutant DNA sequences were generated by site-directed mutagenesis (38) in pBluescript vectors (Stratagene). All mutations were sequenced by the dideoxy method with Sequenase (U. S. Biochemical Corp.).

Bacterial Expression of Proteins

The Myb domain of P (Pmyb) was obtained by introduction of a BamHI site 6 base pairs upstream of the ATG codon of Dp1962 (26). A blunted BamHI-RsaI fragment (the RsaI site is immediately after the Myb domain of P; Ref. 9) was cloned into the blunt-ended BamHI site of pET19b (Invitrogen). The Myb domain of v-Myb (Mybmyb) was obtained by PCR. The 5'-end primer used for the generation of Mybmyb (v-Myb5pET) contained the sequence corresponding to the first 8 amino acids of v-Myb used in other studies (39), in which an ATG codon was introduced, and sequences around it were converted into an NcoI site. In addition, an XhoI site was added 5' to the NcoI site, which added an extra Ser residue upstream of the Met residue. Thus, the N10His-Mybmyb protein contains the amino acids Ser, Met, and Ala between the original v-Myb sequences (39) and the polyhistidine tag present in the pET19b vector (Invitrogen). The 3'-end primer for the cloning of Mybmyb contained the sequence corresponding to the last 8 residues of the Myb domain (residues 123-130 in Ref. 39) with the addition of glycine and threonine residues before the stop codon. Primer v-Myb3pET contains a BamHI site 3' to the stop codon. PCR was done on the pT7Myb plasmid (39) kindly provided by Dr. J. S. Lipsick (Department of Pathology, Stanford University), digested with XhoI and BamHI and cloned into pBluescript KS- (Stratagene). To generate MybP1Myb2, each repeat was independently amplified by PCR. For the generation of P1 (R2 Myb repeat of P), a 5'-primer was used (p5pET), which has the same characteristics as v-Myb5pET but with P sequences (9) instead of v-Myb sequences, and a 3'-primer corresponding to residues 59-64 of P (9) in which -Asp64 was changed to -Glu to create an EcoRV site. Similarly, the second repeat of Myb was amplified using the same 3'-primer as before, but a 5'-end primer corresponding to residues 72-77 (39), which contained a PmlI site, was introduced without any change in the amino acid sequence. The two PCR fragments were purified from agarose, digested with the corresponding enzymes, and ligated, and PCR was then carried out on the ligation mixture with primers p5pET and v-Myb3pET. PCR products were cloned as above.

To generate MybMyb1P2, we first introduced a SacI site in Mybmyb. This was achieved by amplification of each Myb repeat independently, with 5'-primer v-Myb5pET and a 3'-primer targeted to sequences encoding residues 70-76 (39), in which the Val residue at position 77 (39) was changed to Leu, to generate a SacI site. As described above, the fragments corresponding to the two Myb repeats were cut with SacI, ligated, and amplified using v-Myb5pET and v-Myb3pET. This yields a Mybmyb domain with Val77 changed to Leu. The presence of this leucine does not have a significant effect on the DNA binding activity of v-Myb (not shown). In a similar fashion, a Pmyb domain was created in which a SacI site was introduced between R2 and R3 using a 5'-primer for the second Myb repeat of P (R3) containing the SacI site, resulting in the change of Asp64 to Glu and Val65 to Leu (9), and a 3' primer (P3pET) with properties similar to those of v-Myb3pET, which binds DNA as well as the wild type (not shown). This construct was then digested with SacI and BamHI and used to replace the second Myb repeat of Myb in the Mybmyb-containing construct, yielding the chimeric protein MybMyb1P2. To verify that no mutations were introduced by PCR, the inserts were sequenced.

To express the corresponding proteins in bacteria, the XhoI-BamHI inserts were introduced into the corresponding sites from pET19b (Novagen). The clones carrying the right inserts were transformed into the BL21(DE3)PlyS Escherichia coli strain. Protein expression was achieved by induction of cultures (A600 of 0.3-0.5) with 1 mM isopropyl-1-thio-beta -D-galactopyranoside for 2 h at 37 °C.

To generate the change in P of Leu55 to Glu, the first Myb repeat of P was amplified with a 3'-primer containing the sequence corresponding to the last 15 residues of the first Myb repeat of P (9), in which the codon corresponding to Leu55 was replaced by the codon corresponding to Glu, including a SacI site at the 5'-end and at the 5'-end primer P5pET. The fragment was digested with XhoI and SacI and used to replace the corresponding fragment of the Pmyb domain containing the SacI site at the boundary between both Myb repeats.

To generate all other residue substitutions, site-directed mutagenesis (38) of the corresponding Myb domains cloned in pBluescript plasmids was used. All the mutant Myb domains were sequenced before the corresponding fragments were ligated to pET19b for expression in E. coli.

Purification of PolyHis Proteins

Proteins were expressed in 150-ml cultures as described above, harvested by centrifugation, and lysed in 5 ml of SB (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, and 100 µg/ml phenylmethylsulfonyl fluoride) by sonication with a microtip at maximal power for three pulses of 30 s in ice. Samples were centrifuged for 10 min at 12,000 × g and filtered through two layers of Miracloth. RNase A and DNase I were added to a final concentration of 1 mg/liter, and samples were kept on ice for 10 min. 0.5 ml of a 50% slurry of Ni resin (Qiagen) equilibrated with SB was added to each sample and incubated with rocking at 4 °C for 1 h. The resin was loaded onto a column, washed three times with 3 ml of SB, followed by two washes with 1.5 ml of WB (50 mM sodium phosphate, pH 8.0, 300 mM NaCl, 1% Tween 20, 10% glycerol, 5 mM beta -mercaptoethanol, and 10 mM EDTA). Elution was carried out by three sequential additions of 0.5 ml of 100 mM imidazol in WB. Protein levels were estimated by SDS-polyacrylamide gel electrophoresis by Coomassie Blue and silver staining and comparing with immunoglobulin standards. The yield of >90% pure recombinant protein obtained in these conditions is between 0.5 and 5 mg of protein/liter of culture, depending on the particular Myb domain expressed.

Gel Mobility Shift Analysis

Labeling of oligonucleotides for gel mobility shift analysis was carried out as described (26). Cloned fragments were labeled after cleavage with HindIII and SacI and fill-in of the 3'-recessive HindIII site with [alpha -32P]dATP (37). Labeled fragments were purified from native polyacrylamide gels.

DNA binding reaction incubations were carried out as described (26, 39). Probes were used at concentrations between 0.1 and 1 nM. When pure proteins were used, 30-100 ng (about 30-100 nM final concentration) were incubated with the DNA probe. If total E. coli extracts were used instead of pure protein, each binding assay contained the amount corresponding to 20 µl of bacterial extract. Free and bound complexes were resolved by electrophoresis through 1.5-mm-thick 7% polyacrylamide gels (80:1 acrylamide:bisacrylamide) in 0.25 × TBE buffer at 40 V/cm for 55 min at 4 °C, unless otherwise indicated. Apparent Kd values were determined by incubation of fixed amounts of N10His-Pmyb protein with increasing amounts of APB1 probe (Fig. 1C) and determination of the bound and free oligonucleotide concentration by use of a PhosphorImager followed by Scatchard analysis. Studies for a particular protein-DNA complex were carried out independently at least three times.


Fig. 1. DNA binding properties of the Myb domain of P. A, gel mobility retardation experiments with E. coli-expressed P Myb domain as a polyhistidine fusion (N10His-Pmyb) to wild type (APB1, lane 1), mutant oligonucleotides at each (APB2 and APB3, lanes 2 and 3), or both P binding sites (APB5, lane 4) identified in the promoter of the P-regulated gene A1 (26). B, gel mobility retardation analysis with E. coli extracts containing the N10His-Pmyb protein and APB1 as probe. Increasing amounts (200 or 1,000 molar excess) of different competitor DNA were added. The protein-DNA complex and the free probe are indicated. C, sequence of the oligonucleotide probes used for DNA binding studies. I and II, two overlapping P binding sites present in the A1 promoter (26).
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Quantitations were done with a BAS2000 PhosphorImager (Fuji).

Binding Site Selection

The oligonucleotides used for these studies are the same as reported previously and were labeled in a similar way (26). For the first round of selection we made use of the polyhistidine tag present on all the proteins tested by incubating total E. coli extracts containing about 20 ng of the protein of interest with 0.4 ng of degenerate oligonucleotide. Once binding was completed, Ni2+ resin (Qiagen) equilibrated in binding buffer was added to the reaction, which was then incubated for 1 h in ice and washed three times with binding buffer. The resin was resuspended in 20 µl of water, and 1 µl was used for the generation of the probe for the next round by PCR (26). The four subsequent rounds of selection were done by excision of the shifted bands from preparative gel mobility shift assays as described (26) and amplification of the extracted DNA probes. After five rounds of selection, the PCR products were cut with BamHI and EcoRI and cloned into the corresponding sites of pBluescript KS-. Sequences were aligned on the basis of our previous studies of the sequences preferentially bound by P and v-Myb (26).


RESULTS

DNA Binding Properties of the Myb Domain of P

To determine whether the Myb domain is solely responsible for the DNA binding properties of P (26), the Myb domain of P (residues 1-122) was expressed as an amino-terminal polyhistidine fusion protein in E. coli (N10His-Pmyb). DNA binding activity of the fusion protein was then tested on wild-type and mutant forms of the previously identified element in the A1 promoter, which has two overlapping P binding sites (APB1; Ref. 26). Fig. 1A shows that N10His-Pmyb can bind effectively to APB1, and this binding is similar to that of the full-length P protein (26). Point mutations at either site I (APB2; Fig. 1C) or site II (APB3; Fig. 1C) slightly reduce binding (Fig. 1A, lanes 2 and 3, respectively), and binding to a mutant at both sites (APB5; Fig. 1C) was not detected (Fig. 1A, lane 4). Oligonucleotides containing only site I (PBS1; Fig. 1C), or site II (PBS2; Fig. 1C) efficiently compete binding of N10His-Pmyb to APB1 (Fig. 1B, lanes 4-7), as does APB1 itself (Fig. 1B, lanes 2 and 3), whereas oligonucleotides containing the corresponding mutant sites (PBS3 and APB5; Fig. 1C) do not compete the binding of N10His-Pmyb to APB1 (Fig. 1C, lanes 8-10). N10His-Pmyb binds APB1 in vitro with high affinity (Kd, 28 ± 3 nM; data not shown). Binding of the Myb domain of P to APB1 is severely reduced by mutations in the DNA recognition helices of the second or the third P Myb repeats, as well as by addition of monoclonal antibodies specific for each Myb repeat to the binding reaction (not shown). These results indicate that both Myb repeats of P are necessary and sufficient for high affinity sequence-specific DNA binding of P to the binding sites identified in the A1 promoter.

A highly conserved cysteine residue (C130 in c-Myb, C53 in P) has been proposed to act as a molecular sensor in vivo for a REDOX regulatory mechanism for v-Myb and c-Myb (40, 41). We asked whether P requires a reduced environment to bind DNA in vitro, as v-Myb does. To test this, the full-length P and v-Myb proteins were expressed in E. coli as described before (26) and tested for binding on their respective binding sites (APB1 for P and mim-1 wt for v-Myb; Ref. 26) (Note: full-length P forms three shifted complexes, labeled I-III in Fig. 2. Whereas complex I is formed by the intact P protein, complexes II and III were determined to be formed by P proteolytic products (data not shown). Both v-Myb and P bind DNA very poorly, if at all, when DTT is omitted from the binding buffer (Fig. 2, compare lanes 1 and 2 and 11 and 12, respectively). Furthermore, addition of the oxidizing agent diamide, which oxidizes free sulfhydryl groups, prior to the incubation with the DNA completely inhibits binding of both v-Myb and P (Fig. 2, lanes 3 and 13, respectively). This effect is reversed by the addition of excess DTT (Fig. 2, lanes 4 and 14). However, if free sulfhydryl groups are irreversibly alkylated by the addition of N-ethylmaleimide (NEM), protein-DNA complex formation is dramatically reduced in the case of both v-Myb and P (Fig. 2, lanes 5 and 15), and binding cannot be restored by the addition of excess DTT (Fig. 2, lanes 6 and 16). The irreversible effect of NEM on complex formation is only observed when the proteins are in their reduced state. If NEM is added to proteins that have not been reduced previously with DTT, the inhibition by NEM is reversed by the addition of excess DTT (Fig. 2, compare lanes 9 and 10 and 19 and 20). Similar results were obtained using just the Myb domains of P and v-Myb (not shown). These results indicate that P must be in a reduced state to bind DNA, providing the first example of a plant Myb protein with similar REDOX requirements as animal Myb proteins (40, 41, 42).


Fig. 2. P, as v-Myb, requires a reduced REDOX state to bind DNA. Gel mobility retardation experiment with the mim-1 wt probe and total E. coli extracts containing v-Myb (lanes 1-10, v-Myb), or APB1 as probe and total E. coli extracts containing P (lanes 11-20, P). + and -, addition and no addition of particular reagents, respectively before incubation with DNA. The reagents were added in the order shown. The complex formed by v-Myb with the mim-1 wt probe is indicated. Three complexes form when P is incubated with APB1. Complex I corresponds to the full-length P protein, and complexes II and III correspond to specific degradation products, as determined by the use of P monoclonal antibodies directed against different regions of the P protein (E. Grotewold, unpublished results).
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The Myb Domains of P and v-Myb Are Sufficient to Confer Differential DNA Binding Activities to These Proteins

To understand whether the DNA binding properties of P are solely given by its Myb domain, or whether other regions of the protein contributed to sequence-specific DNA binding, we compared the binding specificities of the Myb domain of P with those of the intact P protein. The Myb domains of P and v-Myb were expressed in E. coli as polyhistidine fusion (N10His-Pmyb and N10His-Mybmyb, respectively). As previously determined for the intact P and v-Myb proteins (26), the Myb domain of P does not bind the v-Myb binding sites present in the promoter of the mim-1 gene (mim-1 wt; Fig. 1C), and the Myb domain of v-Myb does not bind the P binding sites of the promoter of the A1 gene (APB1; Fig. 1C and data not shown). Their in vitro DNA binding preferences were tested using a site selection strategy on a random population of 26-mer oligonucleotides, as was described previously (26). Fig. 3 shows the comparison of the sequences selected by N10His-Pmyb and P (Fig. 3, A and B) and by N10His-Mybmyb and by v-Myb (Fig. 3, C and D). Overall, the corresponding Myb domains exhibit DNA binding preferences very similar to those of the intact proteins. These results demonstrate that the Myb domains of P and v-Myb are responsible for their different DNA binding preferences (26), and the contribution of the remainder of the respective proteins to the in vitro DNA binding properties is probably minimal. These results suggest that the highly conserved Myb domains of other plant Myb proteins are also sufficient for sequence-specific DNA binding.


Fig. 3. The DNA binding specificities of P and v-Myb are determined by the corresponding Myb domains and not by other regions of the proteins. Graphic representation of the results of binding site selection experiments, expressed as percentage of times a particular nucleotide was found at each position. The proteins used in each case are indicated on the top of each panel, and the number of sequences analyzed is shown in parentheses. Positions of the nucleotides from -4 to +3 were arbitrarily assigned. Favored residues selected for each position are shown below. N, any residue is accepted. If two residues are accepted, they are shown separated by a shill. If one is favored over the other, the less preferred residue is shown in smaller type. The corresponding shades for each residue are indicated on the bottom right. B and D, previously selected sequences (26) included for comparison purposes. Only sequences containing single binding sites were included in this analysis.
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Analysis of Differences between Plant and Animal Myb Domains

Having determined that the Myb domains of P and v-Myb are sufficient for their distinct DNA binding preferences, we investigated to what extent the differences present between plant and animal Myb domains, represented in P and v-Myb, have a role in DNA binding activity. The high degree of conservation in the DNA recognition helices among animal and plant Myb domains, as well as the differences between these two classes of Myb domain proteins, are illustrated in Fig. 4 (differences marked with dots and arrows). Several previous studies have investigated the effect of mutations of specific residues on the DNA binding activity of v-Myb and c-Myb (30, 31, 34, 43). The uniqueness of this study is that residue substitutions were designed based on the differences between plant and animal Myb domain proteins. Four residues are different in the R2 DNA recognition helices between plant and animal Myb domains (Gln129, Glu132, His135, and Asn137; Fig. 4). We focused our studies on Glu132 of v-Myb, which corresponds to a leucine residue in all plant Myb proteins (Leu55 in P; Fig. 4) and which may play a role in base pair recognition, as well as in cooperative interaction between the two Myb repeats (33). The effect of mutations at Glu132, however, had not been previously studied. We investigated the effect of the change in v-Myb of Glu132 to Leu. The protein was expressed as a polyhistidine fusion (N10His-MybmybE132L) and tested for binding to P and v-Myb binding sites (Fig. 5, A and B). This mutation showed no significant effect on DNA binding to the mim-1 wt probe (Fig. 5A, bottom panel, compare lanes 2 and 3), indicating that its role in stabilizing the cooperative interaction of the two Myb repeats of v-Myb is minor, if significant at all. This mutant protein does not bind to the P binding sites present in the APB1 probe under the conditions tested (Fig. 5A, top panel, lane 3). Site selection experiments carried out with N10His-MybmybE132L did not show any significant binding consensus difference between N10His-MybmybE132L and N10His-Mybmyb (not shown), supporting the idea that Glu132 does not play a role in sequence-specific DNA binding. The corresponding substitution introduced in the Myb domain of P (N10His-PmybL55E) inhibits DNA binding activity of P to the APB1 probe (Fig. 5A, top panel, compare lanes 7 and 8) and does not allow P to bind to the Myb binding sites (Fig. 5A, bottom panel, lane 8). In fact, the effect of this amino acid substitution in P has a more dramatic effect on DNA binding activity than the change of a conserved asparagine residue (Asn109, Fig. 4) to leucine (N10His-PmybN109L) (Fig. 5A, lane 9). Site selection experiments confirmed that N10His-PmybL55E has a general inability to bind DNA, since no sequences could be selected by this protein from a random population of oligonucleotides (not shown). We then investigated the effect of substitutions in the second recognition helix of v-Myb with the corresponding residues in P. Initially, we changed all three residues that differ between P and v-Myb in this DNA recognition helix (Ala180, Val181, and His184; Fig. 4, arrows) to the corresponding residues found in P and most other plant Myb proteins (MybmybAVH-EIY). This substitution resulted in a complete loss of DNA binding activity by v-Myb to the mim-1 wt site (Fig. 5A, bottom panel, lane 4). The substitution of Ala180 in v-Myb with the corresponding Glu found in all plant Myb proteins or the substitution of Val181 and Tyr184 with the corresponding residues of P (Fig. 4) completely abolished binding of v-Myb to mim-1 wt, without a significant gain of DNA binding activity on APB1 (not shown). However, we have not tested whether these residue changes abolish binding of v-Myb only to the mim-1 wt site or whether they also affect the DNA binding activity of v-Myb in a more general way.


Fig. 4. Sequence alignment of several animal and plant Myb domains. The vertebrate Myb domains of mouse c-Myb (16, 51), A-Myb, and B-Myb (18) are compared with v-Myb (36, 51). Plant Myb domains of the maize (Zm) C1 (7), MybZm1 and MybZm38, barley (Hv) MybHv1, Hv33 (8), MybHv5 (52) and GAMyb (14), Arabidopsis (At) GL1 (11), Atmyb6 and Atmyb7 (13), Antirrhinum (Am) MybAm305, MybAm340, MybAm330, MybAm315, MybAm308, and MybAm306 (10), and Petunia (Ph) MybPh1, MybPh2, and MybPh3 (12) proteins are compared with the Myb domain of the maize P protein (9). Dots, identical amino acids with the sequences of v-Myb and P, which are shown in bold. The presumed DNA recognition helices are boxed, amino acid differences between v-Myb and P are marked with dots or arrows over the corresponding residues, and residues mutated in this study are indicated with arrows.
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Fig. 5. Differences between plant and animal Myb domains are important for DNA binding activity. A, gel retardation analysis with 43 ng of pure polyhistidine-tagged (N10His-) Myb domains. The names of the corresponding Myb domains are indicated. Binding was tested on the P (APB1) and Myb (mim-1 wt) binding sites. B, quantitation of DNA binding activity of the different Myb domains as determined by the gel mobility retardation experiments shown in A. The percentage of bound probe (% Bound) was estimated by use of a PhosphorImager and by division of the bound radioactivity by the total radioactivity in each lane.
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We next investigated whether a compensatory substitution in R2 is required for the proteins containing substitutions in R3 to bind DNA. In particular, we found striking the observation that animal Myb domains have the charged Glu132 residue in R2 that corresponds to a leucine residue in plants, whereas most plant Myb domains have a charged residue at a similar position in R3, replaced by a hydrophobic residue in animal Myb domains (Ala180 in v-Myb; Fig. 4). Thus, we combined the E132L substitution with the A180E substitution (N10His-MybmybE132LA180E) and the E132L substitution with the substitution of the three residues different between P and v-Myb in the DNA recognition helix of R3 (N10His-MybmybE132LAVH-EIY). No significant binding to the mim-1 wt or the APB1 probes was detected when these mutants were tested for DNA binding activity (Fig. 5, A, lanes 5 and 6, and B), indicating that the E132L substitution is not sufficient to allow v-Myb proteins containing the residue changes in R3 to bind DNA.

Plant and animal Myb domains also differ in the length of the second Myb repeat (R2); plant Myb domain proteins contain an insertion of a leucine residue between the second and third helices (Fig. 4). We tested the effect of the addition of this leucine residue in the v-Myb DNA binding domain at the position normally found in plant Myb domains (Fig. 4). Once again, the protein was expressed as a polyhistidine fusion in E. coli (N10His-Mybmyb1(leu)) and tested for DNA binding on the APB1 and mim-1 wt probes. Addition of this leucine residue to v-Myb, however, although sufficient to produce a dramatic reduction in binding to the mim-1 wt probe (Fig. 5A, bottom panel, compare lanes 1 and 2), does not allow binding to the APB1 probe to occur (Fig. 5, A and B), indicating that spacing between helices 2 and 3 in R2 of v-Myb is critical for DNA binding to the mim-1 wt sequence.

Generation of Myb Domains with Altered DNA Binding Preferences by Exchange of Myb Repeats between P and v-Myb

The Myb domain can be thought to be composed of two modules, each represented by one Myb repeat. According to the current understanding of the structure of the Myb domain (33), each module (i.e. Myb repeat) would contribute to sequence-specific DNA binding by recognizing half-sites of the binding site (30). Unlike other bipartite or bimodular DNA binding domains (for example, the POU domain; Ref. 44), each Myb repeat does not bind DNA with high affinity by itself, and cooperative interactions between the repeats are required (33). We investigated whether Myb proteins with novel DNA binding specificities could be created by generating chimeric Myb domains with repeats from Myb proteins with different DNA binding properties. For that purpose, we combined R2 of P with R3 of v-Myb (see "Experimental Procedures") (N10His-MybP1Myb2; Fig. 6A). This chimeric Myb domain binds APB1 at least as efficiently as N10His-Pmyb (Fig. 6B, compare lanes 1 and 3). Binding of N10His-MybP1Myb2 to mim-1 wt is also observed under conditions in which N10His-Pmyb binding is undetectable (Fig. 6B, compare lanes 9 and 11), although this binding is weaker than that of N10His-Mybmyb (Fig. 6B, compare lanes 11 and 12). Mutant versions of the corresponding binding sites are not bound by this chimeric Myb domain, indicating that binding is specific (Fig. 6B, lanes 7 and 15).


Fig. 6. Chimeric Myb domains show different binding characteristics from P or v-Myb. A, the structure of the chimeric Myb domains used in this study is indicated by different shading patterns of the repeats derived from P or v-Myb. B, gel mobility retardation analysis performed with 43 ng of purified E. coli-expressed proteins. Probes are shown on top. Lanes 1, 5, 9, and 13 correspond to binding reactions performed with N10His-Pmyb; lanes 2, 6, 10, and 14 with N10His-MybMyb1P2; lanes 3, 7, 11, and 15 with N10His-MybP1Myb2; and lanes 4, 8, 12, and 16 with N10His-Mybmyb. The positions of the protein-DNA complexes are indicated with arrows, and the position of the free probe is indicated. It is not clear why a doublet is observed for the complex formed by N10His-MybP1Myb2 and mim-1 wt (lane 11).
[View Larger Version of this Image (49K GIF file)]


To demonstrate further that the N10His-MybP1Myb2 chimeric Myb domain has DNA binding preferences different from P and v-Myb, site selection experiments were carried out. The percentages of nucleotides in the DNA sequences selected by the chimeric Myb domain are shown in Fig. 7A. The consensus sequence recognized by the chimeric Myb appears to be a composite of P and v-Myb consensus binding sites. This composite binding site does not have an A at position -4, as is preferred by P and its Myb domain (Fig. 3, A and B). The lack of nucleotide preference at that position, however, is reminiscent of the consensus binding site of v-Myb (Fig. 3, C and D). The lack of a marked preference for any nucleotide at position -3 also resembles the v-Myb binding site. At positions -2 and -1, the binding preference of the chimeric Myb domain is very similar to that of v-Myb. In fact, no sequences selected by N10His-MybP1Myb2 contained a T at position -1, in contrast to the sequences selected by P (Fig. 3, A and B) (26). The composite site at positions 0 and +1 (A and C residues, respectively) of the composite site resembles both the P and v-Myb binding sites, which are the same at those two positions. At position +2, N10His-MybP1Myb2 slightly prefers a C residue, similar to the preference seen for P; v-Myb was shown previously to be flexible at position +2 (29). In our experiments, however, v-Myb seems to prefer a G at this site (Fig. 3, C and D). At position +3, the chimeric Myb domain is much like P, showing no particular preference.


Fig. 7. Myb domains obtained by exchange of Myb repeats from different proteins exhibit unique DNA binding preferences. A, graphic representation of the results of binding site selection experiments using the chimeric Myb domain N10His-MybP1Myb2, expressed as the percentage of times a particular nucleotide was found at each position. B, gel mobility retardation experiment with DI8 as probe and total E. coli extracts containing: lane 1, N10His-Pmyb; lane 2, N10His-MybP1Myb2; lane 3, N10His-Mybmyb; and lane 4, N10His-MybmybE132L. Lane 5, probe without any protein addition. C, gel mobility retardation analysis with DI8 as a probe and total E. coli extract containing N10His-MybP1Myb2 protein in the presence of different competitor DNAs (5 pmol) as indicated above. D, Sequence of DI8. The presumed binding site for N10His-MybP1Myb2 is indicated in bold.
[View Larger Version of this Image (37K GIF file)]


These results agree with the current understanding of the way in which the Myb domain contacts DNA. The R3 Myb repeat recognizes positions -1, 0, and +1 in a very sequence-specific fashion, whereas the R2 Myb repeat recognizes position +3 (33, 35). Our results, however, indicate that the influence of the R3 Myb repeat extends to position -4, whereas both the R2 and R3 Myb repeats seem to have some influence on position +2. The strong binding of the chimeric N10His-MybP1Myb2 protein to APB1 is expected, since one of the P binding sites present in APB1 (ACCAACCT; Fig. 1C) matches perfectly the deduced binding consensus for the chimera (Fig. 7A). The weaker binding to mim-1 wt is probably due to the low preference of the chimera for a G residue at position +2.

These results also suggest that there may be DNA sequences that can be bound by the chimeric Myb domain but not by P or v-Myb. One such sequence, DI8, was identified from the clones selected by N10His-MybP1Myb2 (Fig. 7D). DI8 is recognized by N10His-MybP1Myb2 but not by N10His-Mybmyb or N10His-Pmyb (Fig. 7B, lanes 1-3). The binding of N10His-MybP1Myb2 can be specifically competed by wild-type versions of the P or v-Myb binding sites but not by mutant counterparts, indicating the specificity of the binding (Fig. 7C). The N10His-MybmybE132L protein does not bind DI8 (Fig. 7B, lane 4), indicating that the leucine residue found in the P1Myb2 chimera (L55) is not responsible for this new binding specificity of P1Myb2.

When a chimeric protein containing the first Myb repeat of v-Myb and the second Myb repeat of P (N10His-MybMyb1P2; Fig. 6A) was tested in identical conditions, no binding to either APB1 or mim-1 wt was observed (Fig. 6B, lanes 2 and 10). When this protein was tested on site selection experiments, no sequences were selected (not shown). This indicates that N10His-MybMyb1P2 is unable to bind DNA in general, not just the P and v-Myb binding sites. We cannot rule out, however, that the structure of Myb1P2 is affected by the combination of these Myb repeats, explaining its inability to interact with DNA.


DISCUSSION

Our studies were aimed at studying the structure-function relationship of Myb domains separated by over 600 million years of evolution. We used the maize Myb domain protein P and the avian myeloblastosis virus v-Myb protein as models to investigate how similarities and differences between plant and animal Myb domains affect their DNA binding properties and how novel Myb domains can be created by combining regions from these structurally divergent Myb domains. Previous studies showed that the maize Myb domain protein P has a different DNA binding preference from v-Myb and other animal Myb proteins, providing convenient tools to assay the sequence-specific DNA binding activities of these proteins (26).

The Myb domain of P, as well as the one from v-Myb and from most other plant Myb domain proteins, is formed by two HTH motifs, one corresponding to each Myb repeat. Our studies show that both HTH motifs of P are necessary and sufficient for high affinity DNA binding, similar to animal Myb proteins (33, 35) but different from a plant Myb protein containing a single Myb repeat (45). The Myb domain of P is sufficient for sequence-specific DNA binding, and regions outside the Myb-homologous DNA binding domain have little influence on the DNA sequences preferentially bound by P in vitro. Moreover, the Myb domains of P and v-Myb display similar differences in DNA binding preference, as originally demonstrated for the intact proteins (26), indicating that differences within the Myb domain are responsible for the different DNA binding specificities of these two proteins. Our studies indicate that P requires a reduced state to bind to DNA in vitro. This is similar to what has been found with v-Myb and c-Myb, in which a conserved cysteine residue (Cys130 in c-Myb) could function as a molecular sensor for a REDOX regulatory mechanism (33, 40, 41, 42). We do not know whether a REDOX mechanism regulates P function in vivo (26), but it is tempting to speculate that such a mechanism could control the tissue-specific function of other P alleles (46). Alternatively, this could reflect a general requirement of Myb domains to bind DNA and not a specific mechanism that regulates in vivo activity of particular Myb proteins.

Plant and animal Myb domains differ in a number of aspects, which include several residue differences in the DNA recognition helices of R2 and R3 (Fig. 4). We approached this problem by using a strategy fundamentally different from previous studies (30, 31, 34, 43), which consisted of substituting specific residues in the Myb domain of v-Myb for the corresponding residues present in P and other plant Myb domain proteins, to study their effect on DNA binding activity. The bottom line of these experiments is clear. With the exception of Glu132, all the residue substitutions tested (Fig. 4) had a dramatic inhibitory effect on the DNA binding activity of v-Myb to its binding sites. Moreover, none of the single or multiple residue substitutions tested allowed v-Myb to bind to the P binding sites, suggesting that they are not sufficient for altering DNA binding specificity of Myb domains. Perhaps the specificity of DNA binding by P and v-Myb is influenced by regions outside of the DNA recognition helices, as happens in other proteins containing helix-turn-helix motifs (1). Alternatively, unique interactions could be required between the residues that are conserved among plant or animal Myb domain protein family members, respectively, which are different between the two types of Myb domains. We do not know whether the effect of these residue substitutions is the result of a structural change that prevents DNA binding or whether they affect residues involved in the interaction with DNA. In any case, these findings indicate that these residues play fundamental roles in the DNA binding activity of Myb domains, adding fundamental information to previous structural studies (33, 47, 48). Glu132 provided an exception in many aspects. Glu132 is the only residue difference between plant and animal Myb domain DNA recognition helices that the NMR structure of the c-Myb R2-R3 DNA binding domain implicated in possible base pair contact and cooperative interaction between Myb repeats (33). Yet, replacing Glu132 with the leucine found in all plant Myb domains (Fig. 4) has no effect on the sequence-specific binding of v-Myb to DNA, whereas the corresponding change in P of Leu55 to Glu abolishes the DNA binding activity of P. These results indicate that Glu132 is not important in stabilizing the cooperative interaction between R2 and R3 of c-Myb, or in specific base pair contacts, as was proposed previously (33). In addition, these findings provide evidence that the presence of a conserved leucine, and not glutamic, residue at this position in plant Myb domains is fundamental for DNA binding activity.

All plant Myb domain proteins characterized to date contain an additional leucine residue inserted between the first and second helices of the R2 repeat (Fig. 4), which is absent in animal Myb domains. We investigated the significance of the additional leucine residue by adding it to the Myb domain of v-Myb at the equivalent position. The addition of this residue has an inhibitory effect on the DNA binding activity of v-Myb to its binding sites in the mim-1 wt sequence (Fig. 5, A and B). Since this residue is outside the DNA recognition helices, we conclude that the spacing between the helices in plant and animal Myb domains is critical. The three helices in the first Myb repeat of plant proteins are probably packed differently from the corresponding helices of animal Myb domains.

We investigated the possibility of generating Myb domains with novel DNA binding characteristics by combining components of the Myb domains from proteins with different DNA binding activities. The chimera Myb domain P1Myb2, containing the R2 of P and R3 of v-Myb, binds both the P binding sites present in APB1 that are normally bound by P, but not by v-Myb, as well as the Myb binding sites in mim-1 wt, normally bound by v-Myb, but not by P. The results obtained from site selection experiments carried out with the P1Myb2 Myb domain suggest that this chimeric protein recognizes a site that is a composite between the DNA binding consensus determined for v-Myb and the one determined for P but different from either one. Thus, the P1Myb2 protein has a new DNA binding preference. The prediction that the P1Myb2 chimeric Myb domain should bind DNA sequences that are not recognized by either P or v-Myb was fulfilled by the identification of clone DI8. DI8, which contains the core sequence CTTAACTC, is bound with high affinity only by P1Myb2 but not by P or v-Myb. Contrary to what happens with P1Myb2, Myb1P2 containing the first Myb repeat of v-Myb and the second of P has no in vitro DNA binding activity. These results indicate that within certain limitations, new Myb domains with novel DNA binding specificities can be created by combining Myb repeats of proteins with different DNA binding preferences. In addition to the described DNA binding activity, Myb domains can also mediate protein-protein interactions (49, 50). Thus, novel Myb domains generated in this fashion could be used to target transcription factors to genes containing specific promoters and to modify Myb-mediated interactions with other cellular factors, providing useful tools for both basic and applied studies.

In light of the effect of the previously described mutations in the DNA binding activity of v-Myb, it is interesting that the P1Myb2, but not the Myb1P2, Myb domain chimera binds DNA efficiently. The observation that certain combinations of Myb repeats are unable to bind DNA indicates that corresponding Myb repeats of P and v-Myb are not functionally equivalent. Several reports indicate that the carboxyl-terminal region of R2 of animal Myb domains has a disordered structure, which adopts a helical conformation only on binding to DNA (42, 47, 48). We do not know the structure of the corresponding region of the Myb domain of P or other plant Myb proteins, but interestingly, the helical propensity (53) of the carboxyl-terminal portion of R2 from P is significantly higher than the corresponding region of c-Myb and comparable with the DNA recognition helices of the R3 Myb repeat from plants or animals. We can imagine that Myb domains containing the R2 Myb repeat from P (such as P1Myb2) would require less energy to adopt the double helix-turn-helix structure and will have a stronger DNA binding affinity than Myb domains containing the first repeat of v-Myb (such as Myb1P2). Whereas this is certainly the situation for the chimeric proteins tested in this study, intramolecular interactions specific for each type of Myb domain could play additional stabilizing roles in the formation of stable protein-DNA complexes, interactions probably not conserved in the Myb1P2 chimera.

Together, these results provide the first experimental evidence that plant and animal Myb domains have significant structural differences, despite extensive sequence similarity. In addition, they indicate a need to structurally analyze plant Myb domains to understand the evolutionary mechanisms involved in the high conservation of this domain in a large number of plant transcription factors.


FOOTNOTES

*   This work was funded by National Science Foundation Grant MCB-9406233 (to E. G.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§   To whom correspondence should be addressed: Cold Spring Harbor Laboratory, 1 Bungtown Rd., Cold Spring Harbor, NY 11724. Tel.: 516-367-6860; Fax: 516-367-8369; E-mail: grotewol{at}cshl.org.
1    C. Roberts and E. Grotewold, unpublished data.
2    The abbreviations used are: R, repeat; HTH, helix-turn-helix; REDOX, reduced state; PCR, polymerase chain reaction; TBE, Tris borate/EDTA; wt, wild type; DTT, dithiothreitol; NEM, N-ethylmaleimide; SB, sonication buffer; WB, washing buffer.

Acknowledgments

We thank Ben Bowen for helpful discussions throughout this work, Michele Cleary and Leemor Joshua-Tor for help with computer structural analyses, Hong Qian for the helical propensity program, George Tokiwa for help with PhosphorImager analyses, Greg Marconi for technical assistance, and Hong Ma, Andrea Doseff, Joe Colasanti, Pablo Rabinowicz, Leemor Joshua-Tor, and especially Michele Cleary and Patty Springer for comments and critical reading of the manuscript.


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