(Received for publication, January 27, 1997, and in revised form, March 24, 1997)
From the Department of Biochemistry, McGill
University, Montreal, Quebec, H3G 1Y6 Canada
Pax-3 contains two structurally independent
DNA-binding domains, a paired-domain and a homeodomain. Their
functional interdependence has been suggested by the analysis of the
Sp-delayed (Spd) mouse mutant, in which
a glycine to arginine substitution at position 9 of the paired-domain
abrogates DNA binding by both domains. This glycine is located in the
-turn portion of a
-hairpin motif, and the requirement for this
structure was investigated by mutagenesis at this and neighboring
positions. At position 9, only substitution with proline increased DNA
binding by the paired-domain and homeodomain above the level observed
with the Spd arginine mutation, suggesting that the
-turn is necessary for the function of both DNA-binding domains.
Alanine scanning mutagenesis also identified a number of flanking
residues important for DNA binding by both domains, emphasizing the
requirement of the
-hairpin for the interaction of Pax-3 with DNA.
Furthermore, we show that these mutations reduce binding by the
homeodomain at the monomeric level and do not impair dimerization on a
TAAT(N)2ATTA consensus motif. In contrast, the wild-type
paired-domain was found to prevent dimerization on consensus motifs
with 3-base pair spacing of the type TAAT(N)3ATTA.
Importantly, both the deleterious effect of the Spd
mutation on homeodomain DNA binding and the loss of dimerization on
TAAT(N)3ATTA motifs can be transferred to a heterologous
homeodomain from the human phox protein. Moreover, the presence of the
paired-domain affects sequence discrimination within the 3-base pair
spacer in this context. These analyses establish that the
-hairpin
motif is essential for paired-domain and homeodomain DNA binding, and suggest a novel mechanism by which the paired-domain can influence sequence specificity of the homeodomain within the Pax-3
polypeptide.
The paired-domain was originally described as a region of sequence homology between the Drosophila segmentation gene paired and two genes encoded at the gooseberry locus (1), and was subsequently shown to have a DNA-binding function (2). In addition, paired (Prd)1 and the gooseberry gene products contain a second DNA-binding domain, the paired-type homeodomain. Both domains are conserved in the mammalian Pax-3, -4, -6, and -7 genes, while the remaining 5 Pax genes contain only the paired-domain. Over the past several years, the importance of Pax transcription factors in regulating neurogenesis and somitogenesis has been established through the association of specific developmental disorders in both mice and humans with mutations in the Pax-1, Pax-3, and Pax-6 genes (3). In the case of Pax-3, alterations in this gene are responsible for the Splotch (Sp) mouse mutant (4) and human Waardenburg Syndrome (5, 6), both of which are characterized by pigmentary disturbances in the heterozygous state (7). Homozygous Sp embryos die in utero and display neural tube defects, dysgenesis of various neural crest cell lineages, and an absence of limb musculature (7, 8). In addition, chromosomal translocations involving the PAX-3 (9) and PAX-7 (10) genes have been implicated in the etiology of the human solid tumor alveolar rhabdomyosarcoma, consistent with the aberrant expression of some Pax genes in cellular transformation (11-13).
Both genetic and biochemical studies indicate that the paired-domain comprises two independent and interactive subdomains, NH2-terminal and COOH-terminal, that make distinct contributions to sequence recognition (14, 15). For instance, alternative use of a single glutamine codon in the Pax-3 paired-domain leads to the production of isoforms which differ in their ability to utilize the COOH-terminal subdomain for DNA binding (16). Likewise, an alternative splicing event in the PAX-6 gene encodes an isoform which recognizes DNA exclusively through its COOH-terminal subdomain (15). Adding to this diversity in sequence recognition, consensus sequences derived in vitro for the Prd homeodomain contain palindromic recognition motifs of the type TAAT(N)2-3ATTA to which two homeodomains bind cooperatively (17). Furthermore, regulation of the Drosophila even-skipped gene by Prd requires the cooperative binding of the paired-domain and homeodomain to a target sequence that contains juxtaposed recognition motifs for both domains (18). Consequently, although the paired-domain and homeodomain can bind DNA with high affinity when expressed separately, their presence in the same polypeptide allows them to cooperate in DNA recognition. It is not clear, however, if the DNA-binding properties of Pax proteins which contain both domains are simply determined by the sum of their parts or if interactions between the domains influence DNA recognition.
Our previous analysis of the mouse Spd mutation has
suggested that the paired-domain and homeodomain of Pax-3 may
functionally interact (19). In Spd, a single glycine
to arginine substitution at the ninth position of the paired-domain
abrogates DNA binding to both domains. The elucidation of the crystal
structure for the paired-domain of the Drosophila Prd
protein revealed that the glycine residue contributes to a -turn
that joins 2 short anti-parallel
-strands, together forming a
-hairpin motif (20). To determine the specific requirement for the
-turn and to define the role of the
-hairpin motif in paired-domain and homeodomain DNA binding, additional mutagenesis was
carried out at position 9 and the conserved residues that make up this
structure. The current analysis reveals that the integrity of the
-hairpin is essential for the DNA binding activity of both the
paired-domain and homeodomain, confirming our original hypothesis that
the homeodomain does not function as an independent globular structure
within the intact Pax-3 protein. Importantly, sequence recognition by
the homeodomain was found to differ in the presence or absence of the
paired-domain in both Pax-3 and a chimeric protein containing a
homeodomain derived from the human phox protein, indicating that the
paired-domain of Pax-3 might influence the sequence specificity of the
homeodomain in vivo.
The construction of expression
plasmids encoding wild-type Pax-3, Pax-3Spd
(Pax-3R9) and Pax-3prd has been previously described (19). The
wild-type and Spd Pax-3 constructs comprise a
portion of the Pax-3 cDNA that extends from nucleotides
297 to 1801 (21) cloned into the eukaryotic expression vector pMT2,
allowing for expression in COS-7 cells. Both constructs encode the
full-length 479-amino acid Pax-3 polypeptide and differ by the presence
of a glycine (wild-type) or an arginine (Spd)
residue at the ninth position of the paired-domain. The Pax-3
prd construct lacks an XmaI fragment (positions 342-672 in the
Pax-3 cDNA) which encodes amino acids 17-126 of the
Pax-3 polypeptide and includes the first 92 residues of the
paired-domain. Several other point mutations were created using the
polymerase chain reaction with mutagenic oligonucleotide primers that
replace either the codon for glycine at position 9 of the paired-domain
with codons for cysteine, glutamate, and proline (primers Pax-3C9, E9,
and P9 listed below), or that introduce an alanine codon at positions
6-10 of the Pax-3 paired-domain (primers Pax-3A6-A10 listed below).
The mutagenic primer was used in combination with a second
oligonucleotide primer (5
-CTGTCTCCTGGTACCTGCAC-3
, positions 577-558)
and restriction digestion of the resulting polymerase chain reaction
product produced a MscI-KpnI (positions 396 and 563 in the Pax-3 cDNA, respectively) restriction
fragment that could be used to replace the corresponding portion of the
wild-type Pax-3 XmaI fragment present in pBluescript
(Stratagene). After verifying the sequence integrity of polymerase
chain reaction products by dideoxy nucleotide sequencing, the
XmaI cassette was cloned into the unique XmaI
site of the Pax-3
prd expression construct. The sequence of the
oligonucleotides used for mutagenesis is listed below. In each case,
the oligonucleotide is anchored at the 5
end to position 399 of the
Pax-3 cDNA (21) and the mutagenized codon is underlined:
Pax-3C9, 5
-CCAAGGCCGAGTCAACCAGCTCTGCGGAGTA-3
; Pax-3E9, 5
-CCAAGGCCGAGTCAACCAGCTCGAAGGAGTA-3
; Pax-3P9,
5
-CCAAGGCCGAGTCAACCAGCTCCCAGGAG-3
; Pax-3A6,
5
-CCAAGGCCGAGTCGCCCAGCTC-3
; Pax-3A7,
5
-CCAAGGCCGAGTCAACGCGCTCGG-3
; Pax-3A8,
5
-CCAAGGCCGAGTCAACCAGGCCGGAGG-3
; Pax-3A9,
5
-CCAAGGCCGAGTCAACCAGCTCGCAGGAGTA-3
; Pax-3A10,
5
-CCAAGGCCGAGTCAACCAGCTCGGAGCAGTAT-3
.
Expression constructs were made that contain the Pax-3 paired-domain
fused to the homeodomain of human phox by stepwise addition of
Pax-3 cDNA sequence to the pre-existing pCGNphox
expression plasmid (22). The pCGNphox plasmid contains a cDNA
fragment which encodes the 217-amino acid phox polypeptide and a
21-amino acid (MASSYPYDVPDYASLGGPRSM) amino-terminal extension that
contains the influenza virus hemagglutinin A epitope (23). The addition of Pax-3 sequences involved the introduction of wild-type and Spd XmaI fragments into a unique
XmaI site present in the phox cDNA within codons 3-5
(24). Additional Pax-3 sequence could be added to this plasmid using a
KpnI (position 563 in the Pax-3 cDNA) to
PvuII (position 985 in the Pax-3 cDNA)
fragment which encodes amino acids 90-230 of Pax-3. The unique
KpnI site is contained within the Pax-3 XmaI
fragment and the PvuII restriction site encodes residues 12 (Gln) and 13 (Leu) in both the Pax-3 and phox homeodomains. The
resulting plasmids, phoxPDG9 and phoxPDR9, encode polypeptides of 330 amino acids with a predicted molecular mass of approximately 37 kDa. A
final chimeric construct was synthesized that lacks amino acids 17-126
of Pax-3 by removal of the XmaI fragment as described above
for Pax-3prd.
COS-7 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum at 37 °C under 5% CO2. Approximately 1.5 × 106 COS-7 cells were transfected by calcium-phosphate co-precipitation (25) using 20 µg of supercoiled plasmid DNA prepared by CsCl equilibrium density centrifugation. Precipitates were left on cells for 16 h, at which time the cells were washed once with phosphate-buffered saline, and then treated for 1 min with 15% glycerol in 1 × HEPES-buffered saline (2 × HBS is 280 mM NaCl, 10 mM KCl, 1.5 mM Na2HPO4·2H2O, 12 mM dextrose, and 50 mM HEPES, pH 7.05) at 37 °C. Whole cell extracts were prepared 24 h later by sonication in 200 µl of ice-cold buffer containing 20 mM HEPES (pH 7.6), 150 mM NaCl, 0.5 mM dithiothreitol, 0.2 mM EDTA, 0.2 mM EGTA, and protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1 µg/ml leupeptin, 1 µg/ml aprotinin, and 1 µg/ml pepstatin) (19, 26). The level of Pax-3 expression was determined by Western blotting using a polyclonal anti-Pax-3 antibody (19) at a 1:5000 dilution and visualized by enhanced chemiluminescence using a donkey anti-rabbit horseradish peroxidase-conjugated secondary antibody (Amersham). Similarly, the level of protein expression from the pCGN plasmids was determined using a monoclonal anti-HA antibody (12CA5, BAbCO) at a 1:1000 dilution and a sheep anti-mouse horseradish peroxidase-conjugated secondary antibody (Amersham).
Electrophoretic Mobility Shift AnalysisWhole cell extracts were added to 5 fmol of 32P-labeled oligonucleotide in a 20-µl reaction volume containing 10 mM Tris-HCl (pH 7.5), 2 mM MgCl2, 50 mM NaCl, 1 mM dithiothreitol, 1 mM EDTA, and 5% glycerol (27). In addition, when using the P3OPT oligonucleotide, 1 µg each of p(dI·dC)-p(dI·dC) and sonicated salmon sperm DNA was added as a nonspecific competitor, whereas 2 µg of sonicated salmon sperm DNA was added when the P1/2, P2, and P3 oligonucleotides were used. Protein-DNA complexes were allowed to form at 20 °C for 30 min, and were then loaded onto 6% polyacrylamide:bisacrylamide (29:1) gels containing 0.5 × TBE (90 mM Tris-HCl, 90 mM boric acid, 2 mM EDTA, pH 8.3), and electrophoresed at 12 V/cm in the same buffer. Gels were dried under vacuum and exposed to a PhosphorImager screen (Fuji) so that quantitation of radioactivity could be done using a Fuji BAS 2000 PhosphorImaging station and then to Kodak XAR-5 film with an intensifying screen.
Several oligonucleotides were used in electrophoretic mobility shift
assays to detect either paired-domain or homeodomain DNA binding
activity. The P3OPT oligonucleotide was derived from optimal
recognition sequences determined for the Pax-3 paired-domain in
vitro (28, 29) and contains the core sequence
5-[N]11GTCACGCTT[N]8-3
. In addition, to
reduce nonspecific binding to the P3OPT oligonucleotide, a 20-fold
excess of unlabeled P3OPT was added. Oligonucleotides specific for
homeodomain DNA binding were previously identified by the SELEX
procedure (17) and include P1/2
(5
-[N]11TAATTGAGCG[N]8-3
), P2
(5
-[N]11TAATT-GATTA[N]8-3
), and P3
(5
-[N]11TAATTGGATTA[N]8-3
). Oligonucleotides were synthesized to have a recessed 3
end that would
permit end labeling with [
-32P]dATP and Klenow DNA
polymerase upon annealing.
The
Spd mutation involves an arginine for glycine
substitution at the ninth position of the Pax-3 paired-domain (30).
Based on the three-dimensional crystal structure solved for the
paired-domain of the Drosophila Prd protein, this conserved
glycine residue is situated in a -turn (
1, Fig.
1A) that connects two anti-parallel
-strands (
1 and
2, Fig. 1A),
forming a
-hairpin motif at the amino terminus of the protein. This
motif makes three phosphate contacts and also contributes to
stabilizing the adjacent helix-turn-helix motif (
2 and
3, Fig. 1A), and a second
-turn
(
2, Fig. 1A) that occupies the minor groove
(20). Consequently, disruption of the
-turn is expected to have a
profound effect on DNA binding by the paired-domain as we have
previously shown (19). Interestingly, the Spd
mutation was also found to decrease the homeodomain-specific DNA
binding activity of Pax-3 and suggested that the paired-domain and
homeodomain may functionally interact within the full-length protein
(19). An alternative explanation to this surprising relationship is
that the Spd mutation disturbs folding of the Pax-3
polypeptide in a manner that does not reflect normal interactions
within the wild-type protein. Therefore, it was important to
distinguish between a specific structural requirement for Gly-9 in the
-turn and any nonspecific effects attributed to substitution with
arginine. To accomplish this, the glycine residue was independently
replaced with glutamate, cysteine, and proline to evaluate the role of amino acid charge, size, flexibility, and hydrophobicity in the Spd DNA-binding defect (summarized in Fig.
1B).
Wild-type Pax-3 and position 9 mutants were expressed in COS-7 cells
and Pax-3 protein expression was measured by Western blotting using a
Pax-3 specific antiserum (Fig. 1C). This analysis indicated
that similar amounts of Pax-3 protein were present in the whole cell
extracts corresponding to each of the transfectants, and equivalent
amounts of wild-type and mutant proteins were therefore used in
electrophoretic mobility shift assays (EMSAs). Using the paired-domain
specific oligonucleotide P3OPT, it is apparent that the
Spd substitution (R9) leads to a complete loss of
paired-domain DNA binding activity (compare lanes 1 and
2, Fig. 1D). Results from binding studies with
P3OPT are consistent with those previously obtained using the e5
sequence from the Drosophila even-skipped promoter, where a
17-fold reduction in binding was observed (19). Interestingly, mutation
to glutamate also ablates paired-domain DNA binding activity
(lane 3 in Fig. 1D), indicating that the net
charge of the substituted amino acid is not important in this phenotype. Similarly, substitution with cysteine, which contains a
polar but uncharged side chain, abrogates paired-domain DNA binding
activity. Furthermore, since glutamate and cysteine have smaller side
chains than arginine and can adopt fewer conformations, loss of binding
by these mutants suggests that the size of the substituted amino acid
is not an important determinant in the loss of paired-domain DNA
binding in Spd. The only mutant that does form a
detectable complex with the P3OPT oligonucleotide is proline
(lane 5, Fig. 1D), although it is characterized
by an approximately 20-fold lower affinity than wild-type Pax-3
(lane 1, Fig. 1D). Given that proline is the only substitution that may allow the -turn to form (modeling data not
shown), this analysis would suggest that the Spd
mutation acts primarily through the disruption of this structure.
Is this structural defect also the basis for the loss in homeodomain
DNA binding activity? To answer this, the same series of mutants was
analyzed in EMSAs using a consensus oligonucleotide derived for
homeodomains of the paired class (P2) (17). As we have shown
previously, the Spd arginine substitution reduces
binding to the P2 oligonucleotide approximately 20-fold when compared
with wild-type Pax-3 (compare lanes 1 and 2, Fig.
1E). Likewise, both the glutamate (lane 3, Fig.
1E) and cysteine (lane 4, Fig. 1E)
mutations cause an approximately 20-fold reduction in P2 binding.
Substitution with proline resulted in a mutant that retained
approximately 10% of its homeodomain DNA binding activity (lane
5, Fig. 1E). As was observed with P3OPT, the lack of
any correlation between amino acid charge, size, or hydrophobicity with
the defect in P2 binding, and the reduced severity of the proline
substitution indicate that the loss of the -turn is also responsible
for the reduction in homeodomain DNA binding activity.
The -turn is an essential component of the
-hairpin motif (Fig. 1A). Within the
-hairpin motif,
residues 6-10 are perfectly conserved in all paired-domains described
to date which suggests that they are essential for paired-domain
function (Fig. 2A). As a result, these
residues were chosen for independent alanine replacement to further
define the requirement for the
-hairpin in DNA binding by Pax-3. For
this, the last residue in the first
-strand (Asn-6), the three
residues which make up the
-turn (Gln-7, Leu-8, and Gly-9), and the
first residue (Gly-10) of the second
-strand were individually
mutated to alanine (summarized in Fig. 2A). With the
exception of Gln-7, alanine replacement at each position is predicted
to alter the local secondary structure (lower panel in Fig.
2A), and should therefore affect the functionality of the
-hairpin motif. To visualize the role of these amino acids within
the
-hairpin motif and the possible impact of their replacement with
alanine, the location of side chains is shown schematically with
respect to the
-carbon trace in Fig. 2B. The side chains for Asn-6 and Leu-8 are both directed toward the helix-turn-helix structure (HTH), which binds in the adjacent major groove (refer also
to Fig. 1B). Similarly, replacement of the hydrogen R-group of Gly-10 with the methyl R-group of alanine would occur in the interface with the HTH motif and, as a result, the effect of these substitutions may not be confined to the
-hairpin. In contrast, substitution of the Gly-9 and Gln-7 side chains occurs on the solvent
exposed surface of the
-hairpin structure. Consequently, the
individual mutations are likely to affect the overall structure of the
-hairpin in different ways.
Each of the alanine-substituted proteins was expressed in COS-7 cells
and Pax-3 levels were determined by Western blotting, revealing similar
amounts of Pax-3 polypeptide in each whole cell extract (Fig.
2C). Equivalent amounts of Pax-3 protein were then used in
EMSAs with the paired-domain specific oligonucleotide P3OPT (Fig.
2D). In this analysis, the mutants can be categorized into
three groups based on the extent to which they reduce paired-domain DNA
binding. The first, which includes substitutions of Asn-6, Leu-8, and
Gly-9, do not form a detectable complex with the P3OPT oligonucleotide
when compared with wild-type Pax-3 (compare lanes 2,
4, and 5 to lane 1, Fig.
2D). The drastic loss of binding by the Gly-9 mutant further
illustrates the strict requirement for the -turn in paired-domain
function, in agreement with arginine, glutamate, and cysteine
substitutions at that position (Fig. 1). The next category involves
replacement of Gly-10, which leads to only an approximately 8-fold
reduction in P3OPT binding (lane 6, Fig. 2D) and
may reflect the more conservative nature of this substitution in the
second
-strand. Last, substitution of Gln-7 causes no obvious defect
in paired-domain DNA binding activity (lane 3, Fig.
2D), despite the fact that the side chain of Gln-7 makes a
phosphate contact in the paired-domain crystal structure (20),
suggesting that this contact is not essential for docking of the paired
domain on DNA.
The alanine scanning mutants were then tested with the P2
oligonucleotide to determine the impact of these mutations on Pax-3 homeodomain DNA binding activity (Fig. 2E). Alanine
replacement at Asn-6 and Gly-9 decreased binding to the P2
oligonucleotide approximately 20-fold (lanes 2 and
5, Fig. 2E), whereas mutation of Leu-8 causes an
approximately 5-fold reduction in homeodomain DNA binding activity
(lane 4, Fig. 2E) when each is compared with wild-type Pax-3
(lane 1, Fig. 2E). In addition, the Gly-10 mutant which retained partial paired-domain DNA binding activity also has a
higher degree of homeodomain DNA binding activity when compared with
mutations at positions 6, 8, and 9 (compare lane 6 to
lanes 2, 4, and 5 in Fig. 2E),
although it is still 2-fold lower than the wild-type Pax-3 protein
(lane 1 in Fig. 2E). As was observed with P3OPT,
alanine replacement of Gln-7 also displays a wild-type level of
homeodomain DNA binding activity (lane 3 in Fig.
2D). The same results were obtained with an independent
series of whole cell extracts. This analysis reveals that mutations at
other positions within the -hairpin motif of the paired-domain can
cause defects in binding by the Pax-3 homeodomain, and is consistent
with our original hypothesis that the two domains functionally
interact. Importantly, the general correlation between paired-domain
and homeodomain DNA binding properties in this mutagenic analysis suggests that the
-hairpin is essential for the DNA binding activity of both domains.
In the analyses described above, the DNA binding
properties of the Pax-3 homeodomain were investigated using the P2
oligonucleotide as it represents the consensus binding sequence for
paired class homeodomains containing a serine at position 50 (17). In
this case, dimerization by the homeodomain occurs with 30-40-fold
cooperativity over monomeric binding and, accordingly, the predominant
complex formed by the position 9 and alanine scanning mutants appears to be the slower migrating dimeric form (Fig. 1E and Fig.
2E). It was therefore important to distinguish between a
requirement for the -hairpin structure in monomeric binding by the
homeodomain from any effects the disruption of this structure could
have on dimerization. To address this issue, the monomeric homeodomain binding properties of position 9 and alanine-replacement mutants were
characterized using an oligonucleotide which contains a single TAAT
motif (P1/2) (Fig. 3).
The difference in Pax-3 binding to P1/2 and P2 is illustrated in Fig.
3A where the single, lower-affinity complex formed with the
P1/2 oligonucleotide (lane 2) corresponds to the faster
migrating of the two complexes observed with the P2 oligonucleotide
(lane 1). Comparing the P1/2 binding activities of wild-type
Pax-3 to the R9, C9, E9, and P9 mutants (compare lane 3 to
lanes 4-7 in Fig. 3), a clear defect in formation of the
P1/2 complex can be observed. Furthermore, alanine replacement of
Asn-6, Leu-8, Gly-9, and Gly-10, which all exhibited greatly reduced
binding to both P3OPT and P2 (Fig. 2, D and E),
do not interact with the P1/2 oligonucleotide at a detectable level
(lanes 8, 10, 11, and 12 in Fig. 3). On the other
hand, substitution of Gln-7 with alanine did not affect P1/2 binding
activity (lane 9, Fig. 3), consistent with its wild-type
binding properties on P3OPT and P2. These results indicate that
disruption of the -hairpin motif affects monomeric binding by the
Pax-3 homeodomain and does not represent a defect in the ability of the
Pax-3 homeodomain to dimerize.
Is the requirement of the -hairpin structure for
homeodomain DNA-binding a unique property of the Pax-3 homeodomain? To
address this question, a chimera was constructed which fused the Pax-3 paired-domain and linker region to the human phox homeodomain. The phox
homeodomain was chosen as it displays a reasonably high degree of
identity to the Pax-3 homeodomain (67%), yet it normally functions
without a paired-domain (24). Furthermore, the presence of a glutamine
residue at position 50 of the phox homeodomain confers DNA binding
properties that are distinct from those dictated by the serine present
at this location in the Pax-3 homeodomain (17, 31). The chimera
involved the replacement of amino acids 22 to 107 of pCGNphox with a
region comprising amino acids 18-230 from Pax-3 (Fig.
4A). In addition, the pCGN expression
constructs encodes an epitope for hemagglutinin A (HA) at their amino
termini. The wild-type phox and chimeric constructs were expressed in
COS-7 cells and proteins were detected by Western blotting using a
monoclonal anti-HA antibody (Fig. 4B). The size of the
proteins detected by the antibody corresponds to those predicted from
the primary sequence of phox (25 kDa) and the Pax-3 chimeras (37 kDa)
and indicates that the wild-type (phoxPDG9) and Spd
(phoxDPR9) chimeras are expressed at similar levels (Fig.
4B).
The DNA binding characteristics of the wild-type phox and chimeric
proteins were then analyzed by EMSA using the P1/2 oligonucleotide (Fig. 4C). The wild-type phox protein binds to the P1/2
oligonucleotide with high affinity as expected, as does the phoxPDG9
chimera (compare lane 1 to lanes 2 and
3, Fig. 4C). However, the phoxPDR9 chimera, which
harbors the Spd mutation, shows an approximately
20-fold loss in P1/2 binding activity (lanes 4 and
5, in Fig. 4C) when compared with the phoxPDG9 chimera and wild-type phox. Therefore, the requirement for the -hairpin motif at the level of monomer binding can be transferred to
a heterologous homeodomain.
Paired-class homeodomains have been shown to bind to
palindromic TAAT motifs separated by 2- (P2) or 3-base pair spacers
(P3), and the homeodomain of Drosophila Prd binds P2 and P3
sequences with similar affinity and cooperativity (17). To further
characterize the effect of the paired-domain on homeodomain DNA
binding, we monitored the effect of wild-type or mutant paired-domains
on homeodomain binding to P3 in the context of the full-length Pax-3 protein (Fig. 5). Interestingly, EMSAs of wild-type
Pax-3 on the P3 oligonucleotide (lane 3 and 4,
Fig. 5A) only detected the lower affinity monomeric complex
upon comparison to complexes formed with the P2 oligonucleotide
(lanes 1 and 2, Fig. 5A) when using a
fixed amount of Pax-3 and oligonucleotides labeled to the same specific
activity. In addition, the P3 DNA binding properties of position 9 and
alanine scanning mutants (data not shown) were identical to those
observed with the P1/2 oligonucleotide (Fig. 3). To determine if this
inability to dimerize on the P3 oligonucleotide derives from the
presence of the paired-domain or simply represents an inherent property
of the Pax-3 homeodomain, the P3 binding properties of a protein
(Pax-3prd) lacking the first 92 amino acids of the paired-domain
were investigated. Results shown in Fig. 5B indicate that
Pax-3
prd can dimerize to the same extent on P2 (lane 3)
and P3 oligonucleotides (lane 4), whereas intact Pax-3 can
only dimerize on P2 (compare lanes 1 and 2, Fig.
5B). Therefore, the presence of the paired-domain in the
Pax-3 protein precludes dimerization of the homeodomain on the P3
oligonucleotide.
The failure of Pax-3 to dimerize on the P3 oligonucleotide was somewhat
surprising given that in vitro selection of consensus motifs
for the closely related Pax-6 homeodomain revealed its preference for a
P3-based motif (32). The Pax-6 derived P3 oligonucleotide was therefore
used in EMSAs with Pax-3 to determine if its unique sequence
composition affected the ability of Pax-3 to dimerize. Remarkably, this
oligonucleotide shows a complete failure to bind to the Pax-3
homeodomain when compared with P2 (compare lanes 1 and
2, Fig. 5C). This result is even more striking
when using phox, phoxPDG9, and phoxprd (lanes 3-8, Fig.
5C) for the following reason: the presence of glutamine at
position 50 of the phox homeodomain allows it to dimerize with
approximately 10-fold greater cooperativity on P3 motifs than
paired-class homeodomains containing a serine at this position (17,
31). Despite this difference, the phoxPDG9 chimera fails to bind to the
Pax-6 derived P3 oligonucleotide (lane 6, Fig.
5C) when compared with its binding to P2 as a control (lane 3, Fig. 5C). Moreover, the ability of both
the phox and phox
prd proteins to efficiently dimerize on this
oligonucleotide (lanes 7 and 8, Fig.
5C), when compared with their monomeric binding to P2
(lanes 4 and 5, Fig. 5C), indicates
that the presence of the paired-domain is responsible for the failure
of the phox homeodomain to bind the Pax-6-derived P3 sequence
effectively.
The Pax-6-derived oligonucleotide (P3.S in Fig.
5D) differs from the original P3 sequence in several ways:
it only contains 4 base pairs flanking the palindromic homeodomain
recognition site, and both the flanking and spacer sequences differ
from that of P2 and P3 (Fig. 5D). To determine if such
differences impact upon sequence recognition in the presence of the
paired-domain, a series of P3-based oligonucleotides were synthesized
that involved the stepwise replacement of sequences in P3.S with those
present in P3 (P3.3 and P3.5 in Fig.
5D). In this analysis, the phox homeodomain was chosen
because of its greater propensity to dimerize on P3-type sequences.
Accordingly, the predominant complex formed between phox and the P3,
P3.5, and P3.3 oligonucleotides is dimeric (lanes 1-3, Fig.
5E), and the slightly lower affinity of P3.5 and P3.3 can be
attributed to the presence of a non-consensus purine residue following
the TAAT motif on the complementary strand (17). As was observed with
P3.S (lane 6, Fig. 5C), the phoxPDG9 chimera also
fails to bind the P3.5 and P3.3 oligonucleotides (lanes 5 and 6, Fig. 5C). In contrast, phoxPDG9 does bind to the P3
oligonucleotide efficiently (lane 4, Fig. 5E) and
indicates that the differences in the spacer sequence between P3 and
P3.3 can account for the loss of homeodomain DNA-binding. However, the
sequence differences present in these P3 oligonucleotides do not
restore homeodomain dimerization to the phoxPDG9 chimera, and this is
consistent with the behavior of Pax-3 (Fig. 5C and data not
shown). Finally, the restoration of dimerization on P3, P3.3, and P3.5
by deletion of the paired-domain in phoxprd (lanes 7-9,
Fig. 5E) indicates that it is indeed the paired-domain which
confers these additional sequence constraints and prevents homeodomain
dimerization on P3-based sequences.
The paired-domain has been highly conserved throughout evolution
and defines the nine members of the mammalian Pax gene
family (3). Like Prd and the two gene products of the Drosophila
gooseberry locus, the murine Pax-3, -4, -6, and -7 proteins also
contain a homeodomain. In the Caenorhabditis elegans PAX-6
gene, alternative transcript initiation creates two mRNAs that
encode proteins containing both domains, or only the homeodomain (33,
34). Hence, the presence of the paired-domain and homeodomain in
separate polypeptides would indicate that they can function
independently. In addition, when expressed separately, the
paired-domain (20, 28, 35) and homeodomain (17) bind to distinct,
in vitro-derived consensus sequences with high affinity,
allowing the crystallization of the DNA-bound complexes (20, 36), which
would seem to establish their structural independence. Despite this
apparent independence, rescue of paired null mutants
requires the presence of both domains within the same polypeptide and
mutations in either domain cause similar phenotypes (37). Likewise,
mutations in the paired-domain or homeodomain of human PAX-3 both give
rise to Waardenburg Syndrome type I (38), suggesting that each domain
is necessary for normal Pax-3 function. At the biochemical level, this
possibility was suggested by analysis of the mouse Pax-3
Spd mutant in which a glycine-arginine substitution
within the paired-domain abrogates the DNA binding activity of both
domains (19). The current study extends this observation and
establishes that the integrity of the -hairpin motif in the Pax-3
paired-domain is necessary for DNA binding by both the paired-domain
and homeodomain. An important consequence of their presence in the same
polypeptide is that the recognition of sequences that would normally be
bound by the homeodomain is impaired by the presence of the
paired-domain and this is likely to affect sequence recognition by the
Pax-3 homeodomain in vivo.
Crystallization of the paired-domain of Drosophila Prd
identified two globular subdomains, NH2-terminal and
COOH-terminal, each of which contains three -helices that form a
classical HTH motif (20). In addition, the NH2-terminal
subdomain is characterized by a novel
-hairpin motif that comprises
2 anti-parallel
-strands that are separated by a 3-amino acid
-turn, and this motif contributes extensively to the overall docking
of the paired-domain on DNA. The 2
-strands clamp the DNA-phosphate
backbone and stabilize a second
-turn which engages the DNA minor
groove, and the
-hairpin also interacts with the HTH motif that
occupies the major-groove adjacent to the phosphate clamp (Fig.
1A). In Spd, the arginine mutation
replaces a glycine residue that is necessary for the maintenance of the
-turn, and its disruption would most likely affect the function of
the
-hairpin as a whole, accounting for the abrogation of
paired-domain DNA-binding. The present study establishes that replacing
Gly-9 in the Pax-3 paired-domain with amino acids (Arg, Glu, Cys, and
Ala) of different charge, size, flexibility, and hydrophobicity has a
similar phenotypic consequence. The ability of proline to partially
restore paired-domain DNA binding indicates that these mutations act
primarily through disruption of the
-turn. Furthermore, it is also
clear that the defect in homeodomain binding that occurs in association
with the Spd mutation involves disruption of this
structure and does not simply result from the presence of a positively
charged arginine residue. The integrity of the
-turn is therefore
necessary for the activity of both Pax-3 DNA-binding domains and
suggests that homeodomain does not function independently within the
intact protein.
Of the 9 amino acids which make up the -hairpin motif, only those at
positions 6-10 (Asn-Gln-Leu-Gly-Gly) and 12 (Phe) are conserved in all
paired-domains described to date, suggesting that they have a critical
role in paired-domain function. Mutation of Asn-6 was found to severely
impair DNA binding by both the paired-domain and homeodomain and
identifies it as a key residue within the first
-strand. Given that
the Asn-6 side chain makes a phosphate contact and may also contribute
to the stabilization of the adjacent HTH motif (20), the defect in DNA
binding to the paired-domain would reflect the loss of these
interactions in the presence of alanine. Substitution of Leu-8 and
Gly-10 with alanine is also predicted to affect the local secondary
structure, and may disturb the interaction with the HTH motif where
their side chains normally reside (20). Consistent with this
prediction, the Leu-8 mutant has no detectable paired-domain DNA
binding activity, although it does retain a fraction of its homeodomain
DNA binding activity. The Gly-10 substitution exhibits even greater
homeodomain DNA binding activity and detectable binding to the
paired-domain, in keeping with the more conservative nature of alanine
replacement at position 10. Interestingly, substitution of Gln-7 has no
apparent effect on DNA binding by either domain. Although the presence of alanine is not expected to disrupt the secondary structure and would
still allow contact with the phosphate backbone through the peptide
bond amide group, the Gln-7 side chain normally makes an additional
phosphate contact (20). It therefore seems apparent that this contact
is not essential for docking of the paired-domain on DNA, although it
is possible that the Gln-7 mutant may reduce binding to suboptimal
paired-domain recognition motifs where the phosphate contact makes a
greater overall contribution to DNA binding. These data suggest that
the structural requirement at this position is more important than the
phosphate contact for DNA binding. Significantly, the correlation
between paired-domain and homeodomain DNA binding activities indicates
that the integrity of the
-hairpin is important for DNA binding by
both domains, supporting the notion of an underlying functional
interdependence.
The derivation of optimal binding sequences for paired-class homeodomains established that they cooperatively dimerize on TAAT(N)2-3ATTA motifs (17). Consequently, analyses using the P2 oligonucleotide do not resolve the effect of paired-domain mutations on monomer or dimer binding. It was therefore possible that disruption of a specific paired-domain structure could have a deleterious effect on homeodomain dimerization and not on homeodomain DNA binding per se. However, the nature of this requirement was resolved by establishing that mutations which reduce paired-domain DNA binding activity impair monomer binding by the Pax-3 homeodomain (Fig. 3). The transfer of the Spd defect to the phox homeodomain confirms this finding and also establishes that a heterologous homeodomain can be rendered functionally dependent on the paired-domain. In addition, the recent characterization of the Drosophila even-skipped gene has identified a regulatory element that requires the cooperative binding of the paired-domain and homeodomain of Prd to function (18). Although this establishes that the paired-domain and homeodomain can cooperate in DNA binding, our analyses suggest that the two domains cannot function independently, but must cooperate to enable homeodomain DNA binding.
How is this functional dependence expected to affect sequence recognition by the Pax-3 homeodomain? A possible answer comes with the finding that wild-type Pax-3 failed to dimerize on a series of oligonucleotides that comprise a palindromic homeodomain recognition motif with a 3-base pair spacer (P3) (Fig. 5 and data not shown). The fact that deleting the paired-domain restores dimer formation on P3 oligonucleotides is both consistent with previous analyses in which the isolated paired homeodomain exhibited similar cooperativity on P2 and P3 sequences (17), and suggests that the paired-domain precludes dimer formation on P3 motifs. This possibility is made even more likely with the analysis of the phox chimera for the following reason: the presence of glutamine at position 50 of the phox homeodomain enables much higher cooperativity on P3 sequences than serine 50 homeodomains, which typically exhibit only 40-fold cooperativity (17). Despite this enhanced dimerization potential, the presence of the paired-domain in the phox chimera completely prevents dimerization. This behavior is also supported by the results of in vitro oligonucleotide selection using a Prd polypeptide that contained both a paired-domain and homeodomain (39), or the homeodomain alone (17). Although both assays lead to the identification of the P2 sequence, the P3 motif was no longer selected when the paired-domain was present (39). This suggests that P3 motifs may not be efficiently recognized by Pax proteins in vivo, and that the interactions which allow dimerization of intact Pax-3 on P2 sequences must be fundamentally different than those defined in the crystal structure of the Prd homeodomain complexed to a P3 oligonucleotide.
Although the underlying mechanism involved in the cooperative
interaction of the Pax-3 paired-domain and homeodomain remains to be
defined, our findings parallel those involving other homeodomains, in
which interactions with a second DNA-binding domain are required to
confer appropriate target gene specificity. In the case of the yeast
MAT-a1 and MAT-2 proteins, this specificity arises through the
binding of a MAT-
2-derived amphipathic
-helix to the MAT-a1
homeodomain (40). In isolation, the MAT-a1 homeodomain has no specific
DNA binding activity (41). Similarly, a hydrophobic pentapeptide motif
present in certain Hox proteins is required for cooperative interaction
with a second homeodomain-containing protein, Pbx1 (42). Despite each
of these examples being mechanistically different, such functional
interactions invariably confer novel sequence specificity to the
DNA-binding partner, and are likely to play an important role in
selection of target sequences by Pax-3 in vivo.
We thank Michael Gilman for the gift of the pCGNphox expression plasmid, Gary Leveque for technical assistance, and Kyle Vogan for helpful discussions and critical reading of the manuscript.