The Yeast a1 and 2 Homeodomain Proteins Do Not Contribute

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proteins bind to the DNA, in the 2 homeodomain substitutions of residues that are involved in ... Homeodomain proteins have been found in a wide range of.
MOLECULAR AND CELLULAR BIOLOGY, Jan. 1999, p. 585–593 0270-7306/99/$04.0010 Copyright © 1999, American Society for Microbiology. All Rights Reserved.

Vol. 19, No. 1

The Yeast a1 and a2 Homeodomain Proteins Do Not Contribute Equally to Heterodimeric DNA Binding YISHENG JIN,† HUALIN ZHONG,‡

AND

ANDREW K. VERSHON*

Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854-8020 Received 1 June 1998/Returned for modification 3 August 1998/Accepted 29 September 1998

In diploid cells of the yeast Saccharomyces cerevisiae, the a2 and a1 homeodomain proteins bind cooperatively to sites in the promoters of haploid cell-type-specific genes (hsg) to repress their expression. Although both proteins bind to the DNA, in the a2 homeodomain substitutions of residues that are involved in contacting the DNA have little or no effect on repression in vivo or cooperative DNA binding with a1 protein in vitro. This result brings up the question of the contribution of each protein in the heterodimer complex to the DNAbinding affinity and specificity. To determine the requirements for the a1-a2 homeodomain DNA recognition, we systematically introduced single base-pair substitutions in an a1-a2 DNA-binding site and examined their effects on repression in vivo and DNA binding in vitro. Our results show that nearly all substitutions that significantly decrease repression and DNA-binding affinity are at positions which are specifically contacted by either the a2 or a1 protein. Interestingly, an a2 mutant lacking side chains that make base-specific contacts in the major groove is able to discriminate between the wild-type and mutant DNA sites with the same sequence specificity as the wild-type protein. These results suggest that the specificity of a2 DNA binding in complex with a1 does not rely solely on the residues that make base-specific contacts. We have also examined the contribution of the a1 homeodomain to the binding affinity and specificity of the complex. In contrast to the lack of a defective phenotype produced by mutations in the a2 homeodomain, many of the alanine substitutions of residues in the a1 homeodomain have large effects on a1-a2-mediated repression and DNA binding. This result shows that the two proteins do not make equal contributions to the DNA-binding affinity of the complex. affinity and specificity of a2 binding to its target sites (9, 18, 24, 38). The three-dimensional structures of the a2 homeodomain bound to DNA alone and in complex with a1 and Mcm1 have been determined by X-ray crystallography (27, 40, 45). The a2 homeodomain adopts a fold similar to those of other homeodomains, and many of the DNA contacts are also highly conserved with the structures of other homeodomains bound to DNA (19, 25, 26, 30, 44). Although the DNA contacts made by a2 are almost identical in the three crystal structures, there are some minor differences. One difference among the structures is the position of the N-terminal arm, which makes several additional contacts with the DNA in the a1-a2-DNA and a2-Mcm1-DNA ternary complexes that were not apparent in the a2-DNA cocrystal structure (27, 40, 45). There are also several water molecules at the protein-DNA interface in the a1-a2-DNA ternary complex that were not visible in the a2 cocrystal structure (27). The a1 homeodomain folds in a conformation similar to the a2 homeodomain and makes an extensive set of base-specific contacts in the major groove of its own half site. The N-terminal arm of the a1 homeodomain is disordered in the crystal structure. A comparison of the a2 binding sites in both asg and hsg operators yields the same consensus sequence, 59-CATGTA39. These findings suggest that a2 has the same DNA-binding sequence specificities for both sites. However, it has previously been shown that an a2 homeodomain mutant, H3-3A, with alanine substitutions at residues Ser50, Asn51, and Arg54, which make base-specific contacts in the major groove, affects the ability of a2 to bind DNA and repress transcription in complex with Mcm1 but not with a1 (43). It has therefore been proposed that a1 provides the majority of the DNA-binding specificity and affinity for the a1-a2 heterodimer and that contributions by the a2 homeodomain in complex with a1 are

Homeodomain proteins have been found in a wide range of eukaryotic organisms, spanning the spectrum from yeast to humans. These proteins have in common a conserved 60-residue DNA-binding domain and form a large family of transcription factors that play important roles in cell development (15). Although analysis of homeodomain proteins has shown that many of these proteins bind DNA with relatively low sequence specificity in vitro, they often confer highly specific regulatory activities in vivo (7, 10, 20, 35). One mechanism that homeodomain proteins use to achieve their biological specificity in vivo is through interactions with additional factors (4, 33, 41, 46). These protein-protein interactions function to increase the homeodomain DNA-binding affinity and specificity. One example of this type of interaction involves the a2 protein, which determines cell mating type in the yeast Saccharomyces cerevisiae (22). Although the a2 homeodomain protein binds DNA on its own in vitro, it must interact with one of two other proteins to regulate the cell-type-specific gene expression in vivo. In haploid a cells, a2 protein acts in combination with Mcm1, a MADS box protein, to bind DNA as a heterotetramer and repress transcription of a-specific genes (asg) (23, 31). In diploid a/a cells, a2 protein interacts with a1 protein, another homeodomain protein, to bind DNA as a heterodimer to repress transcription of haploid-specific genes (hsg) (9, 16–18). The interaction of a2 with these cofactors helps increase the * Corresponding author. Mailing address: Waksman Institute of Microbiology and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08854-8020. Phone: (732) 4452905. Fax: (732) 445-5735. E-mail: [email protected]. † Present address: Department of Molecular Biology, Genentech, Inc., South San Francisco, California. ‡ Present address: Laboratory of Cell Biology, Rockefeller University, New York, New York. 585

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relaxed in comparison to those when a2 is in complex with Mcm1. To test this model and to determine the contribution of each homeodomain to the DNA-binding specificity and affinity, we constructed a series of base pair substitutions in the a1-a2 DNA-binding site as well as alanine substitutions in the a1 homeodomain. We examined their effects on a1-a2-mediated repression in vivo and DNA-binding affinity in vitro. In general, our results correlate well with the structural analysis of the a1-a2-DNA complex (27). Interestingly, we show that an a2 mutant, which is lacking all of the base-specific contacts in the major groove, has sequence specificity similar to that of the wild-type protein. This result indicates that the phosphate backbone and minor groove contacts play an important role in sequence-specific recognition by the a2 homeodomain. Finally, we show that a1 contributes to the DNA-binding affinity to the complex, but it appears to have relaxed specificity in comparison with a2. MATERIALS AND METHODS Plasmids and strains. The construction of derivatives of pYJ103, a CYC1-lacZ reporter plasmid containing the different hsg operators, and pAV115, a yeast CEN LEU2 plasmid containing a 4.3-kb MATa locus with the wild-type or mutant a2 gene, has been described (43). Plasmid pYJ195, a PT7, His-tagged a2 C-terminal expression vector, was constructed by cloning a PCR-generated NdeIXhoI fragment which contains a sequence encoding six histidine residues followed by a2 residues 123 to 210 into pET21a(1). pAV123, a yeast plasmid similar to pAV115 but containing the MATa locus, was modified to generate plasmid pYJ210. In pYJ210, a silent PstI site was engineered into the a1 gene at codons for homeodomain residues 43 and 44, and the second intron in a1 was deleted. Derivatives of pYJ210 containing mutant a1 genes were constructed by cloning synthetic PstI-XhoI fragments containing the desired mutations into pYJ210. Plasmid pYJ241, a PT7, His-tagged a1 C-terminal expression vector, was constructed by cloning a PCR-generated NdeI-XhoI fragment that contains DNA encoding a1 residues 66 to 126 followed by six histidine residues into pET21a(1). The haploid MATa and diploid a/a and a/a2-H3-3A strains used in the experiments were described previously (43). b-Galactosidase assays. b-Galactosidase assays were performed as described by Keleher et al. (23). b-Galactosidase activity was measured for three independent transformants for each mutant, and the values were averaged. The standard deviations for all values were less than 10%. Protein purification. The a2 proteins used in the DNA-binding assays are C-terminal fragments containing the residues 123 to 210 with six histidine residues fused to the N terminus. The a2 proteins were expressed from plasmid pYJ195 in the BL21(DE3) pLysS strain. The a1 protein used in the experiments presented in Fig. 3 and 4 is the full-length protein with six histidine residues fused to the C terminus and expressed from plasmid pYJ173 in the BL21(DE3) strain. The a1 proteins used in the experiments presented in Fig. 5 are C-terminal fragments containing residues 66 to 126 with six histidine residues fused to the C terminus. Both a2 and a1 proteins were purified to greater than 90% homogeneity on nickel resin columns according to the manufacturer’s protocol (Novagen). EMSAs. DNA probes used in the electrophoretic mobility shift assays (EMSAs) were synthesized by PCR as described previously (21). EMSAs were performed in a buffer containing 20 mM Tris (pH 8.0), 0.1 mM EDTA, 5 mM MgCl2, 10 mg of bovine serum albumin per ml (fraction V), 5% glycerol, 0.1% Nonidet P-40, and 10 mg of sheared salmon sperm DNA per ml. Protein dilutions were made in 50 mM Tris (pH 8.0), 1 mM EDTA, 500 mM NaCl, 10 mM 2-mercaptoethanol, and 10 mg of bovine serum albumin per ml. Five microliters of the a2 dilution and 5 ml of the a1 dilution were added to 40 ml of end-labeled operator fragment diluted in assay buffer, so that the final NaCl concentration was 100 mM. In the protein-free control, 10 ml of protein dilution buffer was added instead of the a2 and a1 proteins. Reaction mixtures were incubated at room temperature for at least 1 h, and then one half of the reaction mixture was loaded onto a 0.53 Tris–borate–EDTA native 6% polyacrylamide gel and electrophoresed at 200 V for 2 h. Dried gels were exposed to phosphor screens, and the images were scanned on a Molecular Dynamics model 425 phosphorimager.

RESULTS A consensus a1-a2 site mediates repression as well as a wild-type site. We have designed a consensus hsg operator based on the sequence alignment of 17 potential a1-a2 binding sites found in the promoters of hsg (6, 12, 14, 28, 29) (Fig. 1A). This site is very similar to the one used in determining the

FIG. 1. A comparison of naturally occurring and synthetic a1-a2 binding haploid-specific gene operators. (A) Sequence alignment of 17 naturally occurring a1-a2 binding sites located upstream of haploid-specific genes and the synthetic a1-a2 binding sites used in the ternary crystal complex (27) and in this study. DNA sequences are written from 59 to 39 (left to right). (B) Comparison of the repression by naturally occurring and synthetic a1-a2 binding sites. Transcription reporter constructs that contain a1-a2 binding sites in the promoter region of the CYC1-lacZ fusion were transformed into a diploid strain for b-galactosidase assays. The fold (3) repression was calculated by comparing the b-galactosidase (b-Gal.) activities from strains that carry a plasmid containing the a1-a2 site with the activity of a plasmid without the a1-a2 site. The levels of repression are shown for a natural site found in the MATa1 promoter, the synthetic site used to determine the crystal structure of the ternary complex (27), and a partially symmetric synthetic consensus site that we have used as our standard.

crystal structure of the a1-a2-DNA ternary complex and differs from it only at bp 2 and 12, positions in which there are no apparent base-specific contacts in the ternary crystal structure (27). To assay whether the consensus site functions as an a1-a2 repressor site in vivo, a reporter plasmid, pYJ103, was constructed by inserting oligonucleotides containing the site between the UAS and TATA sequences of the CYC1-lacZ promoter fusion in pAV73 (42). The presence of an hsg site in this promoter confers repression of lacZ expression that is dependent on both the a1 and a2 proteins (16). pYJ103 and derivatives containing a natural hsg site from the MATa1 promoter and the site used in the ternary crystal complex were individually transformed into an a/a diploid yeast strain and assayed for b-galactosidase activity (Fig. 1B). The consensus hsg operator conferred 80-fold repression of lacZ expression, while the natural site found in the MATa1 promoter and the site used in the crystal structure conferred 50-fold and 70-fold repression, respectively. We conclude that the consensus operator func-

VOL. 19, 1999

FIG. 2. Effects of base pair substitutions in a consensus a1-a2 DNA-binding site. (A) Predicted base-specific contacts to a consensus a1-a2 binding site. The number of each base pair in the site corresponds to the numbering system used in the analysis of the a1-a2-DNA ternary complex (27). Predicted base-specific contacts in the major groove made by a2 (left half site) or a1 (right half site) are shown above the DNA sequence. Minor groove contacts made by the N-terminal arm of the a2 homeodomain are shown below the DNA sequence. Hydrogen bonds are indicated by arrows, and van der Waals interactions are indicated by lines that end in circles. Water-mediated contacts are indicated by the letter w in a circle. Positions on the DNA where the proteins make sugar-phosphate backbone contacts are indicated with the letter p. (B) Effects of substitutions in the a1-a2 binding site on repression of a heterologous promoter. Values in the table are the repression ratios calculated by comparing the b-galactosidase activities in the presence and absence of the a1-a2 site.

tions in vivo at least as well as or better than the natural a1-a2 site from the MATa1 promoter. We have therefore used this site as the wild-type standard in examining the binding characteristics of the a1-a2 complex. Mutations in the a1-a2 site show reduced a1-a2-mediated repression in vivo. To determine the contribution of each base pair in the hsg operator to a1-a2-mediated repression, sites with single base pair substitutions were cloned into the CYC1lacZ reporter promoter and assayed for b-galactosidase activity in wild-type diploid a/a cells (Fig. 2B). The effects of these substitutions were then compared with the DNA contacts made by a2 or a1 homeodomain observed in the crystal structure of the ternary complex (27). The predicted contacts to the consensus a1-a2 site are summarized in Fig. 2A. In general, substitutions that show the largest reduction in repression occur at positions which are specifically contacted by multiple homeodomain residues in the crystal structure. Substitutions at other positions, in which there is only one base-specific contact in the crystal structure, do not have as large an effect on repression. Interestingly, substitutions at positions in which there are no base-specific contacts also have an effect on repression, suggesting that contacts to the phosphate backbone may have a role in sequence-specific recognition. How specific substitutions correlate with the structure of the ternary complex is summarized below. Substitutions at bp 6, 7, and 19 show the strongest effects on repression (in most cases less than 10% activity of the consensus site was observed). In the crystal structure, these base pairs are contacted by multiple homeodomain residues. Changes at these positions apparently disrupt these contacts, which results in the significant loss of repression. It should be noted that positions 7 and 19 are contacted by the Asn51 side chains in the a2 and a1 proteins, respectively. The Asn51 residue is invariant among all the homeodomain proteins, and this side chain makes virtually identical contacts with an adenine in all of the homeodomains whose structures have been determined (19, 25, 27, 30, 44, 45). Our data are consistent with the idea

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that this residue makes a base-specific contact which is essential for homeodomain DNA binding. Substitutions at bp 5 and 20 show moderate effects on repression (10 to 50% activity). In the crystal structure, residue Ser50 of the a2 homeodomain makes a water-mediated hydrogen bond with base T5. It has been shown that in other homeodomain structures, the positions of the water molecules around residue 50 are not rigidly fixed (2). It is possible that substitutions at this position have only a moderate effect on repression and DNA binding, because the water molecules may be repositioned to make partially favorable contacts with the DNA. Residue Arg55 of a1 makes hydrogen bonds to the N7 and O6 atoms of base G20. Substitutions at this position of CG to AT or GC have moderate effects on repression. However, the CG-to-TA substitution produces almost wild-type repression. It is likely that Arg55 may be able to make a similar contact with the N7 group of the adenine. This result also suggests that the contact of Arg55 with the O6 atom of guanine contributes only a small amount to the overall binding affinity and specificity. Substitutions at bp 8 through 12 have a common characteristic, in that changes from A to T or T to A have little effect on repression (.50% activity), whereas changes to G or C have moderate effects on repression (10 to 50% activity). In the crystal structure of the ternary complex, there are base-specific contacts at positions 8, 10, and 11 in the minor groove by the a2 residues Arg4, Gly5, and Arg7. Since AT base pairs and TA base pairs have similar hydrogen bonding potential and geometry in the minor groove, the protein may bind equally well to either base pair at these positions (36). Replacement with G or C, on the other hand, may disrupt the ability to form the appropriate hydrogen bonds and therefore reduce the DNAbinding affinity of the complex. In addition, both the crystal structure analysis and biochemical studies have shown that the a1-a2 dimer bends DNA in this region (27, 37). Changes at these positions to G or C may cause more resistance to DNA curvature, which may lower the binding affinity of the complex and reduce repression. At bp 15 and 17, two of the three substitutions had little or no effect on repression (.50% activity), whereas the third had rather strong effects on repression (about 10% activity). In the crystal structure of the ternary complex, bp 15 and 17 are contacted by a1 residues Ile50 and Met54, respectively, through van der Waals interactions. In general, it is thought that van der Waals contacts are not critical determinants for binding specificity. This would explain why most substitutions at these positions do not affect repression. The substitutions which do cause a large effect on repression, T15G and C17G, are most likely a result of steric hindrance between the protein and DNA. As is shown below, alanine substitutions of residues that are near these bases relieve this steric interference. Although there are no apparent base-specific contacts at bp 3, 16, and 18 in the ternary complex, our data show that there is sequence specificity at these positions. In the crystal structure, residues Leu26, Tyr25, and Arg53 of a2 make contacts to the sugar-phosphate backbone on either side of bp 3. The contact by Arg53 is conserved among almost all homeodomains (3, 19, 25, 27, 30, 44, 45), and it is also involved in a network of contacts with residues Phe24 and Leu26 in the a1-a2-DNA ternary complex. Substitutions at bp 3 may alter the precise positioning of the sugar-phosphate backbone and therefore interfere with this network of protein-protein and protein-DNA contacts. bp 16 and 18 in the a1 half site are involved in a hydrogen-bond network with five water molecules. It has been suggested that the formation of this hydration interface requires a precise local DNA conformation (27).

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The A16T and A18G substitutions at these positions could have affected the DNA conformation and the water molecule network, which may have resulted in the reduced repression. In addition, the position of the methyl group of the thymine substituted at A16 may cause steric interference with residue Ile50 and thus result in reduced repression. Substitutions at bp 2, 4, 13, 14, and 21 have little or no effect on repression (.50% activity). With the exception of position 4, there are no base-specific contacts by either a2 or a1 residues to these base pairs in the crystal structure, which explains why substitutions at these positions do not have an effect on repression. In the crystal structure, the adenine at position 4 is indirectly contacted by the a2 Ser50 residue through a watermediated hydrogen bond. Since substitutions at this position have no effect on repression, it appears that this contact does not play a critical role in the a2 DNA-binding specificity. Alternatively, the position of the water molecule may be flexible so that it is able to make alternative contacts with the substituting base pairs. Some of the substitutions we have made, T10A, T14G, T14A, T15C, A21C, and A21T, increase the level of repression above the level observed for the consensus a1-a2 site. This result indicates that the consensus site used in our studies is not the optimal repressor site for the a1-a2 complex. Interestingly, substitutions with the largest increase in repression are located at positions in which there are no base-specific contacts observed in the crystal structure. It is possible that these changes allow for better contacts with the phosphate backbone or even permit additional contacts with the bases. Alternatively, these changes may relieve some steric interference between the protein and DNA, which would allow better contacts to the adjacent bases, T15 and C20. Mutations in the hsg operator have reduced a1-a2 DNAbinding affinity in vitro. The results shown above indicate that substitutions at positions in which there are base-specific contacts as well as sugar-phosphate backbone contacts affect a1a2-mediated repression in vivo. To correlate our data for in vivo repression with the effects of these substitutions on the DNA-binding affinity of the complex in vitro, we assayed the operator mutants for their binding affinity by EMSAs with purified fragments of the a1 and a2 proteins (Fig. 3). In general, the DNA-binding results agree with the in vivo repression data. For example, the T7A and T19G substitutions, which show less than 10% of wild-type repression in vivo, cause a 30to 50-fold decrease in DNA-binding affinity compared to the wild-type site. Likewise, T5A and C20G, which have moderate effects on repression in vivo, show approximately fivefold decrease in DNA-binding affinity. Substitutions with no effect on repression in vivo (A4G, T15C, and C20T) have essentially wild-type levels of DNA binding. We conclude that the in vitro a1-a2 DNA-binding affinities of these hsg operator mutants correlate well with their in vivo repression activities and that the decreases in the level of repression are a direct result of lower binding affinity for the mutant sites. An a2 mutant, lacking base-specific contacts, has sequence specificity similar to that of the wild-type protein. Substitutions at base pairs which are contacted by a2 have large effects on a1-a2-mediated repression and DNA binding. Although these results would normally be expected, they stand in contrast to our earlier finding that an a2 homeodomain mutant, called a2:H3-3A, with alanine substitutions of residues Ser50, Asn51, and Arg54, has little or no effect on a1-a2-mediated repression and DNA-binding affinity in complex with a1 (43). Since these substitutions effectively remove the side chains that make base-specific contacts in the major groove, this raises the

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FIG. 3. EMSAs of mutant a1-a2 operator sites. Labeled fragments containing either wild-type or mutant a1-a2 sites were assayed for binding in the presence of a constant amount of a1 and dilutions of a2 (residues 123 to 210) ranging by fivefold increments from 3 3 10210 M (lanes 2, 7, 12, 17, 22, 27, 32, and 37) to 2.4 3 10212 M (lanes 5, 10, 15, 20, 25, 30, 35, and 40). Numbers below the substitutions are the fold (3) repression ratios as measured in the b-galactosidase assay described in the legend for Fig. 2.

question of whether the H3-3A mutant is able to distinguish between the wild-type and mutant sites. To address this question, we examined the ability of the a2:H3-3A mutant to repress mutant hsg operators. Derivatives of reporter plasmid pYJ103 containing a1-a2 binding sites with single base pair substitutions at positions 5, 6, or 7 were assayed in a diploid MATa/MATa2:H3-3A strain, in which the a2:H3-3A mutation is substituted for wild-type a2 at the MAT locus (43) (Fig. 4A). The wild-type site shows approximately the same levels of repression in the wild-type (80-fold) and a2:H3-3A mutant (65-fold) strains. Interestingly, substitutions at bp 5, 6, and 7 have the same effects on the levels of repression in both the wild-type and the a2:H3-3A mutant strains. We next examined the affinity of the purified a2:H3-3A protein binding in complex with a1 to the mutant operators by EMSAs (Fig. 4B). The binding affinity of a1-a2:H3-3A to the G6A and T7A mutant sites is about 50-fold weaker than that to the consensus site, while binding to the T5A mutant site is about 5-fold weaker. We conclude that the binding affinity of the a1-a2:H3-3A complex to the mutant sites in vitro correlates well with the level of repression in vivo. These results show that, despite removal of many of the side chains that make base-specific contacts in the major groove, the a2:H3-3A mutant is still able to discriminate among the mutant sites with the same degree of sequence specificity as the wild-type protein. Mutations in the a1 homeodomain produce reduced repression in vivo. The results shown above indicate that the a2 protein binds in complex with the a1 protein with high specificity and affinity to the site even if it is missing side chains that contact the DNA. This result brings up the question of whether

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a1-a2 DNA RECOGNITION

FIG. 4. (A) Effects of substitutions in the a1-a2 binding site on repression by a2 homeodomain mutant a2:H3-3A. The values are the fold (3) repression of the reporter promoter measured by b-galactosidase assay in the diploid wild-type and a/a2:H3-3A strains and were calculated as described in the legend for Fig. 1. (B) EMSA of a1 and a2:H3-3A bound to the a1-a2 operator sites. a2 contains residues 123 to 210. Labeled fragments containing either wild-type or mutant a1-a2 sites were assayed for binding in the presence of a constant amount of a1 and dilutions of a2 ranging by fivefold increments from 6 3 10211 M (lanes 2, 7, 12, and 17) to 4.8 3 10213 M (lanes 5, 10, 15, and 20).

a1-a2-mediated repression and DNA binding would also be unaffected by substitutions in the a1 homeodomain. In the crystal structure of the a1-a2-DNA ternary complex the a1 homeodomain makes an extensive set of base-specific and sugar-phosphate backbone contacts similar to a2 and other homeodomains (27). Five residues in the a1 homeodomain make base-specific contacts in the major groove of DNA (Fig. 2A). To determine the contribution of a1 to the binding specificity and affinity of the a1-a2 heterodimer, we constructed a1 homeodomain mutants with single alanine substitutions and assayed their effects on a1-a2-mediated repression in vivo (Table 1). For comparison, we have made similar substitutions at the same positions in the a2 homeodomain and examined their

TABLE 1. The effects of similar mutations in the a1 and a2 proteins a1 mutant

Repression activity (%)

Wild type Ile50Ala Asn51Ala Arg53Ala Met54Ala Arg55Ala

100 88 6 12 68 18

a2 mutant

Repression activity (%)

Wild type Ser50Ala Asn51Ala Arg53Ala Arg54Ala

100 76 80 95 90

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effects on repression as well. As predicted by the crystal structure and in contrast to what is observed for similar mutations in a2, substitutions at the residues in a1 that contact DNA significantly reduce the level of repression. For example, the invariant Asn51 residues of both proteins make nearly identical hydrogen-bond contacts with an adenine in their half sites (Fig. 2A) (27). An alanine substitution of this residue in a1 has only 6% of wild-type activity, while the same substitution in a2 has 80% of wild-type activity. The Arg53 residue is conserved among almost all homeodomains and makes similar phosphate backbone contacts in all homeodomains in which the threedimensional structures have been determined. Alanine substitution at this residue in a1 has a large effect on the repression, while the same mutation in a2 has virtually no effect on repression. These results show that residues in the a1 homeodomain make a larger contribution to the activity of the complex than residues at the same positions of the a2 homeodomain. Substitutions were made at other positions in the a1 homeodomain to examine their contributions to repression and DNA binding. In most homeodomain proteins, residue 55 of the homeodomain is a Lys which makes a sugar-phosphate backbone contact (3, 19, 25, 27, 30, 44, 45). However, this residue is an Arg in a1, and in the crystal structure of the ternary complex it makes two base-specific contacts to the DNA. Substitution of this side chain causes a significant decrease in repression, which indicates that residue Arg55 plays a critical role in a1 DNA recognition. Residues Ile50 and Met54 make van der Waals contacts with the bases T15 and C17, respectively. However, alanine substitutions at these residues do not have a strong effect on repression. This result suggests that van der Waals contacts by Ile50 and Met54 do not contribute as much to the DNA-binding affinity as the hydrogen-bond contacts by other residues. Although it has been proposed, in some cases, that residue 50 is critical in determining homeodomain DNA-binding specificity, the mutant with an Ala substitution at residue 50 in the a1 homeodomain still retains 88% of the wild-type activity. This result indicates that this residue does not make a major contribution to the a1 homeodomain DNA-binding affinity. To examine directly the effects of substitutions in the a1 homeodomain on DNA-binding affinity, the purified mutant proteins were assayed for their abilities to bind cooperatively with a2 by EMSAs (Fig. 5B). Mutants that have close to wildtype levels of repression in vivo, such as Ile50Ala, have almost wild-type affinity to the site in complex with a2 in vitro. Other mutations that have intermediate (Arg55Ala) and large (Asn51Ala) effects in vivo cause the same relative decreases in vitro. These results show that the loss of repression by the a1 mutants in vivo is mainly due to the decrease in DNA-binding affinity of the complex. Mutations in the a1 homeodomain produce relaxed DNAbinding specificity. We have shown that the protein produced by the a2:H3-3A mutant has DNA-binding specificity similar to that of the wild-type protein (Fig. 4). This result raises the question of whether the proteins produced by a1 mutants also retain the same DNA-binding specificity as the wild-type protein. To address this question, we assayed for the ability of a1 mutants to repress transcription of reporter constructs with mutant a1-a2 binding sites (Table 2). Our results show that alanine substitutions at residues which contact a specific base in the site still retain some sequence preferences at those base pairs. For example, in the crystal structure residue Ile50 of a1 makes a van der Waals contact with T15. An alanine substitution at this residue, which presumably removes this contact, also produces a decrease in repression with the T15G mutant to a level similar to that for the wild type protein. It is likely

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In combination with a1 homeodomain mutants, the a2: H3-3A displays a mutant phenotype. In contrast to the effects observed for a2 homeodomain mutants, similar substitutions in the a1 homeodomain show reduced hsg repression. This result supports a model in which a1 provides the majority of the DNA-binding energy in the a1-a2-DNA complex. If this model is correct, then the a1-a2-DNA complex may be more sensitive to mutations in the a2 homeodomain if the DNA contacts by a1 are weakened. Under these conditions, substitutions in the a2 homeodomain may have an effect on hsg repression. To test this model, the a1 mutants were cotransformed with the hsg reporter plasmid into the haploid a2: H3-3A strain and assayed for b-galactosidase activity (Fig. 5A). In the presence of wild-type a1 the same levels of hsg repression are observed in the wild-type a2 and a2:H3-3A strains. However, in the presence of the a1 mutants, there is a difference between the levels of hsg repression in the two strains. For example, while the a1 Arg53Ala and Arg55Ala mutants show fourfold and sixfold repression in combination with wild-type a2, these same mutants fail to show any repression in the a2:H3-3A strain. The repression activities by a1 mutants Met54Ala and Ile50Ala are also partially reduced in the a2:H3-3A strain. To ensure that these differences were the result of differences in the DNA-binding affinity of the complex, the a1 mutants were tested for their abilities to bind cooperatively with the a2:H3-3A mutant in vitro (Fig. 5C). The binding affinity of each of the a1 mutant–a2:H3-3A complexes is detectably weaker than that of the same a1 mutant in complex with wild-type a2 (Fig. 5B). These results suggest that the a2 side chains make a small contribution to the binding affinity of the complex. FIG. 5. Effects of alanine substitutions in the a1 homeodomain in combination with wild-type a2 and a2:H3-3A. (A) Plasmids that express the wild-type a1 protein or one of the indicated mutants were cotransformed with the hsg reporter plasmid into either a wild-type or a2:H3-3A strain. In the absence of a1, the reporter vector produces 270 U of b-galactosidase activity. In the presence of wild-type a1 the reporter plasmid produces 8 U, giving 34-fold repression of the promoter by the wild-type protein. EMSAs of the a1 mutants with (B) wild-type a2 protein or (C) the a2:H3-3A mutant are shown. a1 contains residues 66 to 123, and a2 contains residues 123 to 210.

that this base-pair substitution sterically interferes with binding by the protein. Interestingly, the Ile50Ala and Met54Ala mutants show increased repression at the A16T and C17G sites. The effects of these mutations in the proteins are not the result of a general increase in the DNA-binding affinity since they do not suppress the effects of base-pair substitutions at other positions (T15G and A18G). It is likely that the amino acid substitutions at the smaller side chain remove the steric interference caused by the base-pair substitutions at positions 16 and 17.

TABLE 2. Repression by a1 mutations of transcription of reporter constructs with mutant a1-a2 sites a1-a2 site

Wild type T15G A16T C17G A18G C20G a

ND, not done.

Repression (2fold) bya: Wild type

Ile50Ala

Met54Ala

Arg55Ala

59 10 19 5 3 37

48 13 87 97 4 ND

54 12 34 70 3 ND

14 ND ND ND ND 61

DISCUSSION The yeast a2 homeodomain protein requires either the Mcm1 or a1 protein to bind with high affinity and specificity to its target sites and regulate the expression of different celltype-specific genes in vivo. The interaction between a2 and its cofactors dictates which sets of target sites are bound and therefore which sets of genes are regulated. How does the interaction with these proteins influence the DNA-binding specificity of a2? In this study we examined the DNA-binding specificity of the a2 protein in complex with a1. A number of potential a1-a2 binding sites have been identified in the promoters of haploid-specific genes, and many show strong sequence similarity at particular positions in the sites (Fig. 1A). The conclusions from our mutagenesis data about the relative importance of each position in the site correlate well with the sequence conservation among the natural sites. In general, positions which are not conserved among the natural sites, such as positions 11, 13, and 14, can accommodate substitutions without large changes in the level of repression. Positions that we have shown are critical for a1-a2 binding and repression, such as bp 6, 7, and 19, are almost invariant among the known or predicted natural binding sites. The sequence conservation at other positions, however, is not as easy to explain. Among the predicted natural sites, positions 16, 17, 20, and 21 are strongly conserved, but we have shown that many of the substitutions at these positions have little or no effect on repression or DNA binding. One possible explanation for this result is that we have made these substitutions in the context of a strong binding site. It is possible that in the context of weaker binding sites, these positions may make a larger relative contribution to a1-a2 binding and would therefore be conserved. Only a few of the natural sites have been shown by genetic

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and biochemical studies to be functional a1-a2 repressor sites (6, 16, 28). The other sites were identified based on their homology with the known sites and therefore may not be functional repressor sites or may only function weakly. For example, the HO(1) and HO(8) sites vary at several positions that we have shown are important for a1-a2 binding and repression. We have tested these sites for repression in the context of the heterologous CYC1-lacZ reporter promoter and found that they only weakly (two- to threefold) repress the promoter (data not shown). Although these sites function only weakly on their own, they may work synergistically with other sites in the promoter to increase the local concentration of the repressor complex at the HO promoter to repress transcription. The effects of mutations in the a2-Mcm1 binding site on repression and DNA binding have also been examined (39, 47). A comparison of substitutions in the a2-Mcm1 and a1-a2 sites shows that many of the mutations have the same effects on both sites. This result suggests that the binding specificity of a2 in complex with Mcm1 is similar to that in complex with a1. However, we did observe several significant differences between the sites. Substitutions at positions T10 and T11 in the a1-a2 site have relatively little or no effect on repression or DNA binding by the a1-a2 complex. In contrast, substitutions at the analogous positions in the a2-Mcm1 binding site have a large effect (less than 5% activity) on a2-Mcm1-mediated repression in vivo (47). One possible explanation for this difference is that in the a2 and a2-Mcm1 structures the Arg7 side chain makes a contact to the base on the top strand at position 10, while in the a1-a2 complex it makes a contact to the base on the bottom strand (27, 40, 45). These different contacts may have different sequence preferences. However, in the a2Mcm1 complex these positions are also contacted by the Mcm1 protein and we have shown that substitutions of these bases have a large effect on DNA binding and transcription regulation of the Mcm1 protein on its own (1). It is most likely that this difference in the specificity of DNA binding to the two sites is primarily due to binding by Mcm1 and not to differences in recognition by a2. A second difference between the two sites is the effects of substitutions at position A8. A substitution of T at this position in the a1-a2 site produces almost wild-type activity (75%), while the analogous substitution in the a2-Mcm1 site produces only 14% of the wild-type activity (39, 47). In the a1-a2-DNA crystal structure, there are base-specific contacts in the minor groove at this position by residues Arg4 and Gly5 in the Nterminal arm of a2 (27). In contrast, only Gly5 makes a basespecific contact at this position in the a2-Mcm1-DNA structure (40). Although one would assume that there are fewer sequence specific requirements at this position in the a2-Mcm1 site than in the a1-a2 site, we found the opposite. It is not clear from the analysis of the structures why there is this difference between the sites. Although there are these subtle differences, our results show that a2 appears to bind to its half site with similar specificities in complex with Mcm1 and in complex with a1. The analysis of a1-a2 binding to the hsg operator suggests two general properties for homeodomain DNA recognition. First, the base-pair specificity of DNA recognition seems to extend beyond the positions that are contacted by homeodomain residues in the crystal structure. Substitutions at almost every position in the operator have at least a moderate effect on repression and DNA-binding affinity. Although there are not direct contacts to the bases at some positions, there are contacts to the sugar-phosphate backbone. One possible explanation for these results is that these backbone contacts may also be sequence dependent. Substitutions at these positions

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591

may affect the precise configuration of the backbone atoms or the overall DNA structure and therefore have an effect on the DNA-binding affinity of the complex. Although substitutions at these positions have a relatively small effect on the DNAbinding activity in vitro, they have a significant effect on repression in vivo. These results may suggest why sites for many homeodomain proteins from higher eukaryotes that appear to have similar DNA-binding affinities in vitro may have different activities in vivo (5, 8, 11, 32, 35). Secondly, in general, homeodomain proteins seem to have relatively low sequence specificity and display a significant degree of tolerance for different DNA sequences. Our results show that the a1 and a2 homeodomains are similar in this regard, since even substitutions at positions in which there are base-specific contacts (4, 8, 10, 20) have only moderate effects on DNA binding and repression. One possible explanation for this observation is that the proteins may be able to form favorable contacts with the substituted base pairs, and therefore changes at those positions do not dramatically affect the binding affinity. The low sequence specificity may be advantageous for the function of homeodomain proteins. Since homeodomain proteins are often involved in many different combinatorial regulatory circuits, the relaxed DNA specificity may allow these proteins to interact with a number of other cofactors to bind different DNA sites and regulate the expression of different genes (22). The results from the mutational analysis of the a1 homeodomain also correlate well with the crystal structure of the a1-a2-DNA ternary complex. Mutations at residues that make hydrogen-bond contacts with the DNA have a stronger effect on repression and DNA binding than mutations at the residues that make van der Waals contacts. These results were expected since van der Waals interactions are long range and are likely to contribute less energy than hydrogen bonds. Compared with the effects of the same mutations in the a2 homeodomain, mutations in a1 have a larger effect on repression. For example, the Asn51Ala and Arg53Ala substitutions in a2 do not appear to have an effect on a1-a2-mediated repression, while the same mutations in the a1 homeodomain show only about 6% activity of the wild-type protein. These results indicate that despite their similar structures and many similar contacts to the DNA, the two homeodomain proteins do not make equivalent contributions to the DNA-binding affinity of the a1-a2 heterodimer. The result that a1 homeodomain mutants show stronger effects in the a2:H3-3A strain than in the wild-type a2 strain also supports the concept that in the a1-a2-DNA complex, a1 provides a large portion of the DNA-binding energy. Although the a2:H3-3A mutant shows the wild-type level of repression in complex with wild-type a1, when in complex with a1 mutants, the a2:H3-3A mutant displays a more pronounced mutant phenotype. This result suggests that residues in a2 which make base-specific contacts do make a contribution to the DNAbinding affinity of the complex but that it is relatively minor compared to the contribution by the analogous side chains in a1. Our result for the a2:H3-3A mutant created a paradox, i.e., mutations at side chains that make contacts in the major groove of the DNA have little effect on repression and DNA binding, whereas substitutions of the base pairs that are contacted by these residues have a strong effect on repression and DNA-binding affinity. One explanation for this discrepancy is that in the a1-a2-DNA complex, these side chains may not make significant contributions to the binding affinity. Although the substitutions with alanine at these positions remove the a2 base-specific contacts in the major groove, the sequence spec-

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ificity and binding energy provided by the sugar-phosphate backbone contacts and the base-specific contacts by the Nterminal arm in the minor groove may be sufficient for a2 to bind DNA with a1 at close to the wild-type level. A similar phenomenon has been observed for the GCN4 bZIP protein, in which alanine substitutions at residues that contact DNA in the cocrystal structure do not affect the protein’s function in vivo (13, 34). However, our finding that base pair substitutions have a strong effect on repression and DNA-binding affinity is rather surprising. There are two possible explanations. First, although the wild type a2 protein may not require these specific contacts to bind DNA, the Ser50, Asn51, and Arg54 side chains may fit into the DNA only when certain base pairs are at these positions. Changes of these base pairs might cause steric interference with the a2 residues which would result in the reduced binding affinity and repression. If this model is correct, one would then expect that mutant proteins, in which the side chains that cause steric interference are effectively removed by the alanine substitutions, would bind with wildtype affinity to the mutant operator sites. We have shown that proteins containing alanine substituted at a1 residues involved in base-specific contacts bind with relaxed specificity to mutant DNA sites. However, our result that the a2:H3-3A protein is able to discriminate among the mutant operators to the same degree as the wild-type protein argues against this possibility for a2. It is likely that the contacts by the N-terminal arm contribute to some of the sequence specificity of binding by the a2:H3-3A mutant. Another explanation is that changes of these base pairs may have altered the DNA conformation and therefore affected other protein-DNA contacts. For example, it is possible that many of the protein contacts to the sugarphosphate backbone are sequence specific. Therefore, substitutions in the DNA will not only interfere with contacts to the bases but also alter the positions of the atoms in the backbone and thus weaken the protein-DNA contacts. In this case, the crystal structure of the a1–a2:H3-3A dimer bound to the hsg operators is needed to solve the paradox. ACKNOWLEDGMENTS We thank C. Wolberger for providing valuable comments about the a1-a2-DNA crystal structure. We also thank J. Mead for providing plasmid pJM130 and V. Gailus-Durner for comments on the manuscript. This work was supported by a grant from NIH (GM49265) to A.K.V. Y.J. was supported by the Charles and Johanna Busch predoctoral fellowship.

9. 10. 11. 12. 13.

14. 15. 16. 17. 18. 19. 20. 21. 22.

23. 24. 25.

26. 27. 28.

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