Download DNA Specificity of the Bicoid Activator Protein Is Determined by

Cell, Vol. 57, 1275-1283,
June
30, 1989, Copyright
0 1989 by Cell Press
DNA Specificity of the Bicoid Activator Protein
Is Determined by Homeodomain
Recognition Helix
Residue 9
Steven D. Hanes and Roger Brent
Department of Molecular Biology
Massachusetts General Hospital
Boston, Massachusetts 02114 and
Department of Genetics
Harvard Medical School
Boston, Massachusetts 02115
Summary
Formation of anterior structures in the Drosophila embryo requires the product of the gene bicoid. The bi.
coid protein contains a homeodomain
and may exert
its effects in early development
by regulating
transcription of the gap gene, hunchback
(hb). Consistent
with this view, we have demonstrated
that DNA-bound
Bicoid fusion proteins stimulate gene expression.
We
used the gene activation phenotype
in yeast to study
DNA recognition
by the Bicoid homeodomain.
We
found that a single amino acid replacement
at position
9 of the recognition
helix was sufficient to switch the
DNA specificity of the Bicoid protein. The altered specificity Bicoid mutants recognized
DNA sites bound by
Ulfrabithorax,
fushi taram, and other related homeodomain proteins. Our results suggest that DNA specificity in Bicoid and Anfennapedia
class proteins is determined by recognition
helix residue 9.
Introduction
Pattern formation in Drosophila embryos appears to be
initiated by a small number of maternal-effect genes. One
of these genes, bicoid, is necessary for establishing
anterior-posterior
polarity (Ntisslein-Volhard
et al., 1987).
Mutants that lack functional bicoid product fail to develop
head and thoracic structures. bicoid mRNA is synthesized
in nurse cells during oogenesis and transported into the
developing oocyte where it is localized in the anterior (Berleth et al., 1988). After egg deposition, bicoid mRNA is
translated and the protein (here called Bicoid) accumulates in the anterior of the egg (Driever and NtissleinVolhard, 1988a). Bicoid is present in embryonic nuclei
where it may stimulate zygotic expression of hunchback
(Driever and Niisslein-Volhard,
1988b, 1989), and other
gap genes whose expression is necessary to establish
pre-segmental domains in the immature egg (reviewed in
Ingham, 1988).
Like many other proteins important for development, Bicoid contains a conserved 60 amino acid sequence
known as the homeodomain
(reviewed in Gehring, 1987).
Sequence similarity of the homeodomain to the helix-turnhelix DNA binding motif of many bacterial proteins (Pabo
and Sauer, 1984), suggests that the homeodomain
may
also bind DNA (Laughon and Scott, 1984). Indeed, many
Drosophila homeodomain
proteins bind to specific DNA
sequences in vitro (Desplan et al., 1985; Hoey and Levine,
1988; Beachy et al., 1988; Driever and Ntisslein-Volhard,
1989); in some cases, DNA binding has been shown to
require the homeodomain
(Desplan et al., 1988; Miller
et al., 1988b; Hoey et al., 1988). Similarly, some mammalian transcription factors also contain homeodomain
sequences (Scheidereit et al., 1988; Mtiller et al., 1988a)
that are involved in DNA binding (Ko et al., 1988; Sturm
and Herr, 1988).
Many bacteriophage repressor proteins use the second
a-helix of the helix-turn-helix
motif (the recognition helix)
to contact DNA. Sequence specificity is determined primarily by hydrogen bonding of side chains of residues 1
and 2 of the recognition helix to functional groups of base
pairs in the major groove (Lewis et al., 1983; Wharton and
Ptashne, 1985; Hochschild and Ptashne, 1986; Wharton
and Ptashne, 1987; Jordan and Pabo, 1988). Additional
contributions to specificity are made by polar interactions
or hydrogen bonds by residues 5 and 6 (as in h repressor,
Hochschild et al., 1986; or as in 434 repressor, Wharton
and Ptashne, 1987; Aggarwal et al., 1988). Studies of h
and 434 repressor-DNA cocrystals reveal hydrogen bonds
between the amino-terminal
residues of the two helices in
the motif that may help position the recognition helix correctly in the major groove (Aggarwal et al., 1988; Jordan
and Pabo, 1988). Several nonspecific contacts with the
sugar-phosphate
backbone, including one made by the
amino acid at recognition helix position 9, may contribute
to the stability of the repressor-operator
complex.
The amino acid sequence of the Bicoid homeodomain
differs from that in proteins of the Anfennapedia (An@) sequence class (Scott et al., 1989). Several of these differences lie in positions which, by analogy with the bacterial
helix-turn-helix
proteins, might be important for DNA recognition. Amino acids 1 and 2 of the putative Bicoid recognition helix (homeodomain helix 3) differ from those found
at equivalent positions in Antp class proteins, and might
be predicted to make site-specific contact with DNA.
Other residues that are important in bacteriophage
repressors and that differ between Bicoid and Antp class
homeodomain proteins include recognition helix residue
9, which could make nonspecific DNA contacts, and a residue
near
the
amino
terminus
of the
first
helix
of the
motif
(Bicoid residue 127). The position of residue 127 suggests
that it might interact with the first residue in the recognition
helix,
and
thus
help
position
the
recognition
helix
for
favorable contact with DNA.
Bicoid binds to variants of a 9 bp consensus sequence,
TCTAATCCC, that are found upstream of the transcription
start site of the zygotically expressed form of hunchback
(Driever and Niisslein-Volhard,
1989). This hunchback element (or Bicoid binding site) is distinct from sites bound
by other Drosophila homeodomain
proteins. These sites,
TCAATTAAAT (Desplan et al., 1988) and (TAA)” (Beachy
et al., 1988) are bound by proteins encoded by engrailed,
even-skipped, and the Antp class genes Ultrabithorax, Abdominal-A, and fushi tarazu (Desplan et al., 1988; Hoey
and Levine, 1988; Beachyetal.,
1988; Karsch and Bender,
Cdl
1276
personal communication;
Samson et al., 1989). These
sites are not recognized by the Bicoid protein (Samson et
al., 1989; this paper). It seemed possible that Bicoid and
other homeodomain
proteins distinguish between these
sites using residues in the recognition helix which differ,
and are in positions corresponding
to those important for
repressor-DNA recognition.
In this study, we employed fusion proteins that contain
the DNA binding domains of the bacterial LexA repressor
or the yeast GAL4 activator (Brent and Ptashne, 1985; Ma
and Ptashne, 1987a). Whenever examined, fusion proteins that carry a DNA binding domain from the native protein in addtion to the LexA or GAL4 domain stimulate expression of target genes that carry binding sites for the
native protein (Brent and Ptashne, 1985; Hope and Struhl,
1988; Fitzpatrick and Ingles, 1989; Samson et al., 1989).
We took advantage of this fact to examine gene regulation
and DNA binding by Bicoid.
Here we show that DNA-bound LexA-Bicoid and GAL4Bicoid fusion proteins stimulate gene expression in yeast.
Bicoid fusion proteins activated target genes that carried
upstream LexA operators, GAL4 sites, or Bicoid binding
sites. We used the gene activation phenotype to establish
which residues in the Bicoid homeodomain
are required
for DNA recognition. Unlike bacterial helix-turn-helix
proteins, amino acids in positions 1,2, or 5 of the putative recognition helix do not appear to be important. Instead,
“helix swap” experiments showed that Bicoid’s DNA specificity is determined by recognition helix amino acid 9. Our
results also suggest that for Antp class and other closely
related homeoproteins, DNA specificity inheres in residue 9.
Results
DNA-Bound
Bicoid Activates Gene Expression
We tested whether Bicoid regulates gene expression by
producing, in yeast, Bicoid fusion proteins that contained
the DNA binding domains of either LexA or GALA Hybrid
genes were constructed by fusing a bicoid cDNA (Berleth
et al., 1988) to sequences encoding the amino-terminal 87
residues of LexA or the amino-terminal
147 residues of
GAL4 (Figure 1). The fusion proteins were expressed from
an ADHl promoter on 2 urn-based plasmids carrying the
yeast HIS3 gene. All but the first two amino acids of Bicoid
were included in the fusions. To test whether DNA-bound
Bicoid activates gene expression, yeast cells were cotransformed with “maker” plasmids that expressed one of
the fusion proteins, and “target” plasmids that contained
a 2 urn replicator, the yeast MA3 gene, and a GALl-IacZ
reporter gene that carried an appropriate
binding site
positioned upstream of the transcription start site (Figure
2). Changes in GAL&IacZ expression were measured by
assaying 8-galactosidase
activity of cells grown in liquid
culture.
Bicoid fusion proteins stimulated gene expression in
yeast (Tables la and lb). Target genes lacking upstream
binding sites were not stimulated by Bicoid fusion proteins, while those carrying a LexA operator were stimulated about 50-fold by LexA-Bicoid, and those carrying a
GAL4 binding site were stimulated about 20-fold by GALC
a
ADHI
promoter
LexA (I-
/
.
/.
f
Blcold
ima
cDNA
I
TAG
BamHl (Hhal)
.
. .
EcoR’
I 1
Sail
b
GAL4Bicoid
Figure
[I
1. Expression
147
of Hybrid
3
bicoid
489
Proteins
in Yeast
(A) Plasmid pSHP-I
encodes the amino-terminal
87 amino acids of
LexA under control of the alcohol dehydrogenase
I (ADHl) promoter.
Fusions to a bicoidcDNA
were made at the BamHl cloning site. Other
unique sites are EcoRl and Sall. The pSHP-1 vector backbone
and
polylinker
sequence
were derived from pMA424 (Ma and Ptashne,
1987b). The GAU-bicoidfusion
construct
was made at the BamHl site
of pMA424. The DNA sequences
and encoded
amino acids of the
LexA-polylinker-bicoid
fusion junction, and the GAL4-polylinker-bicoid
fusion junction are given in Experimental
Procedures.
(B) Schematic
of the LexA-Bicoid
and GAU-Bicoid
hybrid proteins.
The shaded areas show the approximate
location of the homeodomain. Numbers refer to the amino acid residues in the native proteins.
Bicoid. We interpret these increases in activity of the
reporter gene to reflect increases in transcription due to
DNA-bound proteins (Guarente and Ptashne, 1981; Yocum
et al., 1984). The strong transcription activator LexA-GAL4
stimulated gene expression about 15-fold better than
LexA-Bicoid, and native GAL4 stimulated gene expression about 8-fold better than GAL6Bicoid.
Bicoid Activates Genes Containing
Upstream
hunchbacksequences
To test whether the Bicoid moiety of the LexA- and GALC
fusion proteins recognized specific DNA sites in vivo to activate gene expression, we used sequences found upstream of hunchback that are protected by purified Bicoid
in a DNAase I footprint assay (Driever and NiissleinVolhard, 1989). Multiple copies of this TCTAATCCC consensus Bicoid binding site were inserted upstream of
GALWacZ (Figure 2). Target genes bearing only two copies of the site were not activated by Bicoid fusion proteins
rJJ;;pected
DNA Contact
I
/
I
by Bicoid
Recognition
TATA
,
Helix
GAL I
LacZ
Table
1. Gene
Maker
C-167)
Activation
Plasmids
Target
a
LexA Op
GTACTGTATGTACATACAGTAC
ALexA
<l
<l
<l
LexA(l-a7)
LexA-Bicoid
LexA-GAL4
b
AGAL
GAL+-atv)
TCTAATCCC(TA)
Bicoid site
Antp class site
TCAATTAAATCGA)
Figure
2. Binding
Sites
Used
in This
Study
Autonomously
replicating target plasmids contained the indicated sequences
inserted at a unique Xhol site upstream
of reporter gene
GALMacZ.
Bases in parentheses
were included in these sites, but are
not an integral part of the core sequences.
The LexA operator and
GAL4 binding site were present in single copy, the hunchback
(hb) element was present in 10 copies, and the Antp class binding site was
present in 12 copies. The target plasmids were derivatives
of pLR1 Al
(West et al., 1984) except for the GAL4 site target plasmid, which was
a derivative of pLR1 A20 (West et al., 1984). The Xhol sites in pLR1 At
and pLRlA20
are positioned at 167 bp and 128 bp upstream of the
GALMacZ
transcription
start site, respectively.
(data not shown), whereas target genes bearing ten copies of the Bicoid site were activated more than 500-fold by
LexA-Bicoid and more than 300-fold by GALCBicoid (Tables la and lb). Bicoid target genes were not activated by
the amino termini of LexA or GAL4 alone. Since both fusion proteins activated a Bicoid target, we presumed that
Bicoid site recognition depended on some region common to both proteins, possibly the Bicoid homeodomain.
Helix 3 in the Bicoid Homeodomain
Is Required
for DNA Recognition
Amino acid substitutions were made within the putative
recognition helix of the Bicoid homeodomain
at positions
we thought were likely to make base-specific contacts with
DNA. To destroy DNA recognition, these residues were
changed to alanines, small residues that do not hydrogen
bond to base pairs in DNA, and have been shown not to
disrupt the recognition helices of bacteriophage
repressors (Hochschild et al., 1986; Wharton and Ptashne, 1987;
Hochschild and Ptashne, 1986). By analogy with repressor proteins, the Bicoid recognition helix has been predicted to encompass, minimally, residues 138-147 in the
native protein. Here, these ten amino acids will be referred
to as positions 1 through 10.
To confirm that the mutant LexA-Bicoid proteins were
produced in yeast, we tested their ability to stimulate gene
expression of LexA operator-containing
targets (Table lc).
Only one mutant protein did not satisfy this criterion (mutant A,As&A,);
we assume this protein was unstable or
defective in its activation function. As expected, none of
the mutant proteins activated expression of target genes
that did not contain upstream binding sites.
The mutant proteins were then assayed for activation of
target
genes that carried Bicoid binding sites (Table lc).
C
LexA-Bicoid
LexA-Bicoid
LexA-Bicoid
LexA-Bicoid
A,
As
As
A1A5A.&
Op
Site
ALexA
<I
<l
<l
<l
Bicoid
Fusion
Proteins
Plasmids
<l
<l
<l
GAL+-147)
GALcBicoid
GAL4 site
CGGAAGACTCTCCTCCG
by DNA-Bound
Op
LexA Op
<l
64
844
GAL4
Site
7
19
158
LexA
Op
65
38
94
4
Bicoid Site
<l
564
Bicoid
Site
1
365
1
Bicoid Site
585
187
<l
<l
Results are given in units of 8-galactosidase
activity. Yeast cell cultures cotransformed
with “maker” and “target” plasmids were grown
to a density of 0.8-2.0
x IO’ cells per ml in glucose-supplemented
complete minimal medium lacking uracil and histidine. LexA-Bicoid de
rivatives
were tested in yeast strain MGLD4-4a;
GALCBicoid
derivatives
were tested in a gal- strain, RBY52 (Agal4) in glucosesupplemented
medium. 8-galactosidase
assays were performed on at
least three individual transformants
of a given plasmid combination
(activities varied up to 20%). Assays performed on different days were
normalized
to values obtained for LexA-GAL4
with a LexA operator
target plasmid. LexAo..sr) includes the first 87 amino acids of LexA
plus additional residues encoded by the pSHP-1 polylinker (RPEFPGIRRPAAKLIPAEFLMIYDFYY).
GAL4rr-143 includes the first 147 amino acids of GAL4 plus additional residues encoded by the pMA424
polylinker (PEFPGIRRPAAKLIPAEFLMIYDFYY).
Target plasmids were
all derivatives
of pLRlA1 except for the GAL4 site target, which was
a derivative of pLRlA20.
LexA-Bicoid mutant A, contained a Thr-Ala substitution
at position 1 and still activated a Bicoid site target. LexABicoid mutant A5 contained a Lys+Ala substitution at position 5 and also activated a Bicoid site target but at reduced levels. However, activation by this mutant was also
reduced using a LexA site target, perhaps because the
protein is less stable or impaired in its activation function.
Finally, a LexA-Bicoid mutant that contained a Lys-Ala
substitution at position 9 did not activate a Bicoid site target. This mutant still activated LexA site targets. These experiments indicate that position 9 of the Bicoid recognition
helix is critical for recognition of DNA. The data do not,
however, distinguish between specific and nonspecific interactions with DNA.
Altered Specificity
Bicoid Mutants
To identify those residues in Bicoid required for DNA specificity, we carried out “helix swap” experiments similar to
those described by Wharton and Ptashne (1985). In these
experiments, we changed the predicted solvent exposed
residues in the Bicoid recognition helix to those found in
the recognition helix of Anfennapedia class proteins (Figure 3). The mutant proteins were tested for their ability to
distinguish between two sites known to bind different
classes of homeodomain proteins in vitro. As before, our
assay measured expression of target genes bearing these
upstream sites. Bicoid target genes contained 10 copies
of the hunchback element (TCTAATCCC), and Antp class
Cell
1270
Activation
UQ
I23456789
IO
Antp class
site
Wild-type
Bicoid
+
+
-
AAQVKIWFKN
Al
+
+
-
TAQVAIWFKN
AS
+
+
-
TAQVAIWFAN
As
+
-
RHZ-E I RzQ9
+
-
+
E 1R2Qg
+
-
+
+
+
-
+
-
+
+
-
+
Al AsA6Aq
EBQVKlWFgN
~IEBQVKI
3. Schematic
Bicoid
sil?
TAQVKIWFKN
AAQVAAWFAN
Figure
of Gene Expression
EIRZ
WFKNO
TAQVKIWFQN
Qs
ERQ
I KIWFQN
Antp
Showing
Amino
Acid Residues
class*
in the Putative
Recognition
Helix (helix
3) of Wild-Type
LexA-Bicoid
and Mutant
Derivatives
The corresponding
residues
of Uhbithorax
are shown for comparison,
and are representive
of the consensus
sequence
for Antp class
homeoproteins.
The barrels identify the putative helix-turn-helix
motif of these proteins. The residues that differ from wild-type LexA-Bicoid sequence
are indicated. Activation data are summarized
from Tables 1 and 2 and our unpublished
work. (‘) Results derived from Samson et al. (1989) and
Fitzpatrick
and lngles (1989).
target genes contained 12 copies of an Antp class binding
site (TCAATTAAATGA).
Results are given in Table 2. In the yeast strain used
here, wild-type LexA-Bicoid and the various mutant proteins stimulated expression of LexA target genes to comparable levels, although these levels were uniformly lower
than those obtained previously (see legend to Table 2). As
expected, the mutant proteins did not stimulate target
genes lacking upstream binding sites. Wild-type LexABicoid activated Bicoid site targets but did not activate
Anto class target8 above background levels. LexA-Bicoid
Table
Maker
2. Gene
Activation
Plasmids
Lex$87)
LexA-Bicoid
LexA-Bicoid
LexA-Bicoid
LexA-Bicoid
LexA-Bicoid
LexA-Bicoid
b-‘S
R&g
- El R&g
- E,RP - - - Qs
- - - A9
by Mutant
Bicoid
mutant ErRa, which contains a Thr+Glu substitution at
position 1 and an Ala+Arg substitution at position 2, retained its ability to stimulate expression of target genes
containing Bicoid sites and did not stimulate target genes
containing Art@ class sites. However, LexA-Bicoid mutant
Rk2 -ElR2Q9 which contained, in addition to the changes
at positions 1 and 2, a Leu+Arg substitution at position
3 in helix 2 and a Lys+Gln substitution at position 9, lost
the ability to stimulate target genes carrying Bicoid sites
but gained the ability to stimulate target genes carrying
An@ class sites. A LexA-Bicoid mutant bearing only three
Derivatives
Target
Plasmids
ALexA
<l
<l
Op
LexA Op
<1
6
Bicoid Site
<l
530
Antp
Class
35
26
Site
ALexA
<l
<
<l
<l
<l
Op
LexA Op
7
11
5
8
6
Bicoid Site
<I
<l
750
<l
<l
Antp Class
610
970
25
650
35
Site
Results are given in units of 8-galactosidase
activity. Experimental
conditions are described in the legend to Table 1. Target plasmids (see Figure
2) were all pLR1 Al derivatives
and produced no detectable background
activity, except for the Antp class binding site target plasmid which regularly produced
about 30 U of background
6-galactosidase
activity. In these experiments
yeast strain YM709 was used throughout
because of very
high background
levels of 8-galactosidase
activity produced in strain MGLD4-4a
when transformed
with the Antp site target plasmid.
Unexpected DNA Contact by Bicoid Recognition Helix
1279
of these changes, Elf+&,
exhibited a similar phenotype.
This mutant also stimulated expression of target genes
that carried the Anfp type l(TAA), site (data not shown)
recognized by Ulfrabifhorax protein and other Antp class
proteins (Beachy et al., 1988; Samson et al., 1989). The
results above implicate the importance of residue 9 in
DNA specificity. This was tested directly by constructing
LexA-Bicoid mutant Q,, which bears a single amino acid
substitution of Lys*Gln at position 9 in the recognition helix. This mutant did not stimulate expression of Bicoid site
targets but strongly stimulated expression of targets carrying Antp class sites. For each mutant bearing this
Lys-Gln substitution at postion 9, levels of activation of
Bicoid targets were decreased more than 500-fold, while
levels of activation of Antp class targets were increased
more than 20-fold (Table 2). If one subtracts background
levels obtained with the Antp class target (~30 units), then
postition 9 mutants activated Antp class targets more than
800-fold relative to wild-type LexA-Bicoid.
Discussion
Bicoid Is a DNA Binding-Dependent
Gene Activator
Bicoid is a gene activator. LexA-Bicoid and GAL4-Bicoid
activated expression of target genes that contained LexA
binding sites or GAL4 binding sites, and did not stimulate
gene expression from target genes that lacked binding
sites. The fusion proteins also activated expression of targets genes carrying Bicoid binding sites. Since, in yeast,
LexA sites and GAL4 sites are only bound by proteins that
contain the cognate binding domains, we conclude that
stimulation of gene expression by these Blcoid fusion proteins requires their binding to DNA (Brent and Ptashne,
1985; Giniger et al., 1985; Keegan et al., 1988). These
results support the notion that activation of targets with Bicoid sites in our yeast system and in Drosophila cells
(Driever and Nijsslein-Volhard,
1989) is a consequence of
binding by Bicoid to these sites.
We do not know how Bicoid turns on transcription.
Bicoid contains an acidic region (residues 345-390,
net charge -9), a glutamine-rich
region (or M-repeat,
McGinnis et. al., 1984), and a region that contains alternating histidines and prolines (or PRD repeat, Frigerio et
al., 1988). Acidic regions, glutamine-rich
regions, and
proline-rich regions have all been shown to function as
transcription activation domains (Hope and Struhl, 1988;
Ma and Ptashne, 1987a, 1987b; Courey and Tjian, 1988;
Williams et al., 1988). We observed activation by a GAL4Bicoid derivative that contained only Bicoid residues
3-251. This protein lacked the acidic region and the polyglutamine tract but retained the PRD repeat. It is possible,
therefore, that transcription activation by native Bicoid is
due to the PRD repeat. Alternatively, it is possible that
gene activation by this Bicoid derivative is due to phosphate groups. Bicoid is likely to be differentially
phosphorylated during development
(Driever and NiissleinVolhard, 1989); it is possible that the phosphate groups
may subsitute for acidic amino acids by providing equiva-
lent negative surface charge
and Pelham, 1988).
(Lech et al., 1988; Sorger
Oligomerization
of Bicoid
The DNA bound form of LexA-containing
proteins is a
dimer (Brent and Ptashne, 1981, Brent, 1982). As previously noted for other fusion proteins, the amino terminus
of LexA does not dimerize efficiently, and the fact that
LexA-Bicoid recognizes LexA operators suggests that the
Bicoid moiety contributes to dimerization
(Brent and
Ptashne, 1985; Hope and Struhl, 1988; Lech et al., 1988;
Samson et al., 1989). Whether dimerization occurs on or
off the DNA, the results with LexA-Bicoid thus suggest
that in Drosophila embryos, native Bicoid binds DNA
as an oligomer. Our data suggest Bicoid oligomers may
exhibit another form of cooperativity.
In yeast, Bicoid
strongly stimulated targets that carried ten hunchback elements, but did not detectably stimulate targets that carried
two hunchback elements. This result, which is similar to
results observed with a number of homeodomain proteins
in transient expression experiments (Desplan et al., 1988;
Jaynes and O’Farrell, 1988; Driever and Niisslein-Volhard,
1989), suggests that either binding to the hunchback elements or gene activation by the DNA-bound proteins is
highly cooperative. LexA-Bicoid dimers activate a single
LexA site target more strongly than they activate a target
with two Bicoid sites, which suggests that the cooperativity facilitates binding to the Bicoid sites rather than gene
activation once the protein is bound to DNA. Clusters of
Bicoid binding sites occur upstream of hunchback (Berleth et al., 1988); cooperative binding of Bicoid oligomers
to these sites provides a mechanism by which the continuous Bicoid gradient might be converted into the threecell-wide boundary observed for hunchback expression
(Tautz, 1988).
Unexpected
DNA Contact by the Bicoid
Recognition
Helix
While nothing so simple as a code has emerged, a large
body of work in recent years has suggested that interaction of helix-turn-helix
proteins with DNA is governed by
some common rules (for review, see Pabo and Sauer,
1984; Ptashne, 1988; Schleif, 1988). Protein monomers
typically associate into dimers, in which each symmetrically disposed monomeric subunit interacts with symmetric or near symmetric operator half-sites that lie in adjacent major grooves. In the case of lambdoid phage
repressors and cro proteins, and the E. coli CAP protein,
the second a-helix of the helix-turn-helix motif (the recognition helix) protrudes into the major groove of DNA, and
the amino-terminal
end of the helix makes base-specific
contacts. Sequence alignments (Laughon and Scott, 1984;
Scott et al., 1989) and NMR studies (Otting et al., 1988)
have suggested that Drosophila homeodomain
proteins
contain a helix-turn-helix
motif. If the Bicoid homeodomain interacts with DNA, as do bacteriophage
repressors,
we reasoned that base-specific contacts would be made
by amino acids 1 and 2 of the recognition helix, with possible contributions from residues at positions 5, 6, and 9.
Cell
1280
To test this idea, we made a series of mutant proteins
in which residues in the putative Bicoid recognition helix
were replaced by alanines. The LexA-Bicoid mutants were
examined for their ability to stimulate expression of target
genes that carried upstream Bicoid binding sites. These
experiments benefitted from the fact that the Bicoid derivatives were bifunctional for DNA recognition. By comparing activation of target genes that carried LexA sites or Bicoid sites with those of “wild-type” LexA-Bicoid, we could
ascertain whether a given mutation affected Bicoid DNA
specificity, or affected some other property of the protein
(such as nuclear transport or protein stability). Results indicated that positions 1, 2, and 5 were not important for
recognition of Bicoid sites. However, a mutant that borealanine at position 9 no longer activated Bicoid targets.
These experiments suggested that residue 9 in the recognition helix is important for either specific or nonspecific
interaction with DNA.
To probe the function of individual amino acids in sitespecific DNA recognition, we performed “helix swap” experiments (Wharton et al., 1984; Wharton and Ptashne,
1985). First, we established that Bicoid does not recognize
An@ class sites and that proteins of the Antp class genes,
Ultrabirhorax and Abdominal-A, do not recognize Bicoid
sites (this paper; Samson et al., 1989). Based on these
facts, we attempted to change the DNA specificity of Bicoid by replacing amino acids in its recognition helix with
those found at equivalent positions in the Antp class proteins. A series of LexA-Bicoid derivatives that contained
changes at recognition helix residues 1, 2 and 9, and Bicoid residue 127 (in helix 2) were tested for recognition
of Bicoid and Antp class sites. Proteins that contained
changes at positions 1 and 2 still recognized Bicoid sites,
whereas all proteins with changes that included a LysGln substitution at position 9 no longer recognized Bicoid
sites, but recognized Antp class sites. Most dramatically,
a mutant protein in which the only change was a Lys-+Gln
substitution at residue 9 exhibited a complete switch of
specificity; it did not recognize Bicoid sites but did recognize Antp class sites.
These results show that Lys at position 9 of the recognition helix is critical for DNA specificity by Bicoid. A single
Lys+Gln change, in the context of a Bicoid recognition helix, confers Antp class DNA recognition on the Bicoid protein. This implies that Gln at position 9 is also critical for
DNA specificity by Antennapedia
class proteins and that
Bicoid and Antp class proteins contact DNA in an analogous manner. The simplest interpretation is that, unlike
the bacteriophage
repressor proteins, amino acid 9 of Bicoid and Antp class recognition
helices make basespecific contacts with DNA, while residues at the the
amino terminus of the recognition helix do not. This model
suggests that the recognition helix of these homeodomain
proteins is oriented differently in the major groove than the
recognition helix of bacteriophage
repressor proteins.
It is not difficult to imagine how changing a single lysine
in Bicoid’s position 9 would result in a dramatic change in
specificity. Most Drosophila homeodomain
proteins, including Antp class and related proteins encoded by the
genes engrailed, even-skipped, and zerkniillt, contain a
glutamine at this position (Scott et al., 1989) and recognize the type 1 (TAA)” and type 2 (TCAATTAAAT) sites
used here (Beachy et al., 1988; Beachy, personal communication; Karsch and Bender, personal communication;
Desplan et al., 1988; Hoey and Levine, 1988; Samson et
al., 1989). It is possible that the side-chain amide of the
Gln donates a hydrogen bond to N7 of an Adenine in the
type 1 and type 2 sites, and accepts a hydrogen bond from
N8 of the same base (Seeman et al., 1978). Bicoid has a
Lys at position 9; and by analogy with models proposed
for h cro protein, it is possible that the amino group of the
Lys donates hydrogen bonds to the 06 and N7 of a Guanine in ?he Bicoid site (Ohlendorf et al., 1982). In this view,
each LexA-Bicoid monomer that carries a Gln-+Lys substitution at position 9 loses two hydrogen bonds to the Bicoid
site and gains two hydrogen bonds to the Antp class sites,
a difference in energy sufficient to account for the altered
DNA specificity of LexA-Bicoid with Gln at position 9.
Based on the identity of position 9, there are at least
three other classes of homeodomain
proteins: proteins
such as paired product, which has a Ser at this position,
proteins such as cut product, which has His at this position, and POU class proteins, which have Cys at this position (Frigerio et al., 1986; Blochlinger et al., 1988; Finney
et al., 1988; Herr et al., 1988). If these proteins contact
DNA like the proteins studied here, then all members of
a class should bind similar sequences, members of different classes should recognize different sequences, and in
each case some of the DNA specificity should derive from
contacts between residue 9 and the binding site.
Experimental
Procedures
Strains, Media, and p-Galactosidase
Assays
Escherichia
coli strains were JMIOI (supE thi A(/ac-proAB)
[PtraDSB
pra4B lacP ZAM75]), BW313 (HfrKL16 PO/45 [/ysA(61-62)]
dutl ungl
tbil m/Al), CSH50 (A(/ac-pro)
thi ara s&I/f
(proA
Mac/q ZAM75
traD36), BMH 71-18 (A[/ac-proAs]
thi supE F’/ac/q ZAM75 proAB),
BMH 71-18 mutS (A[/ac-proM]
thi supE F’lacilqAM75
proAB
mutS275:;TnlO),
MK30-3 (A[/ac-pfa46]
mcd ga/E strA Wacis ZAM75
proAB). Saccharomyces
cerevisiae
strains used were MGLD4-4a
(a
ura3-52 leu2 his3 trp7 lys2 cyrr), RBY52 (a Dgal4 ura3-52 leu2 hisd),
and YM709 (a ura3-52 his3-200 ade2-707 /ys2-807 trp7-907 tyrl-507
met canR ga/4-542 ga/80-538).
Bacteriological
work was done using
standard techniques
(Miller, 1972; Ausubel et al., 1987). Yeast media
preparation and cell growth were carried out as described
in Sherman
et al. (1978). Yeast transformations
were done using the LiOAc procedure (Ito et al., 1983). fi-galactosidase
assays of liquid cultures were
carried out as described
by Harshman
(1988) using at least three individual yeast transformants.
Cells were grown in complete minimal
medium (lacking uracil and histidine) to mid-logarithmic
stage and one
ml was collected by centrifugation.
The cell pellet was resuspended
in
200 ~1 of 0.1 M I-is-HCI (pH 75), 0.05% Triton X-160 and frozen at -800 C.
After thawing slowly on ice, B-galactosidase
assays were carried out
on permeablized
cells, and cell debris was then removed by centrifugation. Units are expressed
as (lOOO)(&~)/(minutes
of reaction)(cell
volume)(OD&
and varied up to about 20% between individual isolates.
Plasmid Construction
DNA manipulations
were carried out by standard methods (Maniatis
et al., 1982; Ausubel et al., 1967). Oligonucleotides
were synthesized
by the phosphate triester method using a Biosearch Series 8ooO DNA
Synthesizer
and purified using Applied Biosystems
oligonucleotide
purification
cartridges.
A GAL4-fusion
plasmid, pMA424 (Ma and Ptashne, 1987b) was ob-
Unexpected
1281
DNA Contact
by Bicoid
Recognition
Helix
tained from Jun Ma. A LexA-fusion
plasmid, pSHP-1, was constructed
by insertion of an EcoRI-Sacl(*3
kb) fragment of pMA424 containing
a polylinker,
ADHl terminator,
and yeast 2 pm replicator
into the
EcoRI-Sac1
backbone of pMA457 (Ma and Ptashne, 1987b). The ADHl
promoter-LexA
fragment
in pMA457
was originally
derived
from
pRBlO27 (Brent and Ptashne, 1985). The second BamHl site upstream
of the ADHl promoter
in this plasmid (pSHI-1)
was then destroyed
creating pSHP-I.
Plasmid pSH2-1 is essentially
identical to pMA424
except that the DNA binding region of GAL4 (residues l-147) has been
replaced by the DNA binding region of LexA (residues i-87). Both plasmids contain the HIS3 gene for selection in yeast. Unique sites in these
plasmids for fusions to LexA or GAL4 are EcoRI, BamHI, and Sail. The
sequence
of the polylinker
from EcoRl to Sall is WTTCCCGGGGATCCGTCGAC.
The reading frame in the polylinker
in each of the
plasmids (pSH2-1; pMA424) is the same, and is indicated by the underline. Fusion of a nearly full length bicoid cDNA (Berleth et al., 1988)
to LexAo-rr,) and GAL4r1-t4r) was accomplished
in several steps. First,
the cDNA (in EcoRl site of a Bluescript vector) was digested with Hhal,
made flush with T4 DNA polymerase,
and then digested with Sall. The
743 bp fragment was then ligated to pMA424 that had been previously
digested with BamHI, Klenow treated, and Sall digested. The resulting
plasmid was then digested with Sall, and the remainder of the bicoid
cDNA sequence
(including more than 1 kb of 3’ flanking sequences)
was inserted as a Sall-Sal1 (ml.8 kb) fragment.
The final plasmid,
pSHlO-1, encodes an in-frame fusion of GAL4 ,,-,,r, to residue 3 (Gln)
of the bicoid coding sequence
separated
by an 18 bp polylinker
sequence. A BamHI-Sac1
fragment containing bicoid and about 2.5 kb
of adjacent vector sequences
was removed and inserted
into the
BamHI-Sac1
backbone
of pSHP-1. The resulting plasmid, pSHll-1.
contains an in-frame fusion of LexA(I-sr, to residue 3 of Bicoid via a 21
bp linker. The top strand sequence across the junction, and the corresponding amino acid sequence of the LexABicoid
fusion is as follows:
CCT(LexAsr)-CGACCGGAATTCCCGGGGATC-CAA(Bicoid&
P-RPEFPGI-Q.
The top strand sequence
across the junction, and corresponding
amino acid sequence
of the GAL4Bicoid
fusion is as follows: TCG(GAL4&-CCGGAATTCCCGGGGATC-CAA(Bicoid&
S-PEFPGI-G.
The fusion junctions were confirmed
by DNA sequence
analysis.
All target plasmids (except pSV15) were derivatives
of LRI Al (West
et al., 1985) and contain the yeast URA3 gene, 2 urn replicator, and a
GALI-/acZfusion
gene downstream
of a GAL7 promoter from which the
UASo had been removed. Plasmid ~I840 (Brent and Ptashne, 1985)
contains a single 22 bp LexA operator (Brent and Ptashne, 1984) in the
Xhol site at -167 bp from the GALI-/acZ
transcription
start site. pSV15
is an LRl A20 derivative (West et al., 1984) and contains a 17 bp GAL4
consensus
binding site (Giniger et al., 1985) in the Xhol site at approximately -128 bp from the GAD/acZ
transcription
start site. LRl A20 is
otherwise identical to LRl Al except that the Xhol site is positioned 39
bp closer to the transcription
start site. Target plasmids containing multiple copies of the hunchback
consensus
sequence
TCTAATCCC
(Driever and Niisslein-Volhard,
1989) were constructed
by ligation of
complementary
28 bp oligonucleotides
of sequence TCGAATCTAATCCCTATCTAATCCCT
and TCGAAGGGATTAGATAGGGATTAGAT
into
the Xhol site (-167) of LRl Al. Plasmid pHB9 contains five 26 bp
hunchback
oligonucleotide
inserts (ten consensus
binding sites).
Target plasmids containing multiple copies of the Anfp class binding
site consensus
sequenceTCAATTAAAlGA(Desplan
et al., 1988) were
constructed
by ligation of complementary
54 bp oligonucleotides
of
sequence
TCGAGTCAATTAAATGATCAATTAAATGATCAATTAAATGATCAAT TAAATGAC and TCGAGTCATTTAATTGATCATTTAATTGATCATTTAATTGATCATTTAATTGAC
into the Xhol site (-167) of LRll.
Plasmid pFtz-7 contains three 54 bp Antp sequence class binding site
oligonucleotide
inserts (12 consensus
binding sites).
Mutagenesis
of Bicoid Helix 3
Oligonucleotidedirected
mutagenesis
was carried out by the methods
of Kunkel et al. (1987) or Kramer et al. (1984). For creation of the A,
and EIRP mutants (see Figure 3). the bicoid-containing
EcoRl fragment of pSHll-1
was removed and inserted into the EcoRl site of
phagmid pUC119 (Vieira and Messing,
1987). DNA was transformed
into strain E. coli strain BW313 (dufl ungl) and single-stranded
DNA
prepared (after superinfection
with phage M13K07) from recombinants
containing bicoid inserts in the desired orientation.
Plus strand muta-
genie oligonucleotides
were treated with T4 polynucleotide
kinase, annealed in excess to bicoid template DNAs (minus strands),
and the
mixtures
extended with T4 polymerase
and ligated according
to the
method of Kunkel et al. (1987) as described
in Ausubel et al. (1987). The
DNA was then transformed
into CSH50 cells (dut+ ung+) for selection
of mutant plasmids. Initial screening was for a newly created restriction
site (88111) introduced
by the mutant oligonucleotides.
The sequences
of the oligonucleotides
used for creating the A, and E,Rz mutations
were GCCCTGGGCGCAGCCCAGGTGAAGATCTGGTTTAAGAACCG
and CTAGCCCTGGGCGAACGCCAGGTGAAGATCTGGTTTAAGAACCG, respectively.
The bases changed from wild-type are underlined.
For creation of mutants As, As, AtA&&,,
Rh2-E,R.&,
E,RzQs, and
09 (Figure 3) the method of Kramer et al. (1984) was employed using
commercially
available
reagents
(Boehringer
Mannheim).
bicoidcontaining EcoRl fragments from the LexA-Bicoid expression
plasmids
were subcloned
into phage M13mp9 am 16 and propagated
in strain
BMH 71-18 (supE). Single-stranded
DNA of minus strand polarity (with
respect to bicoid sequences)
was annealed to linearized M13mp9 rev
86 and to excess
mutagenic
oligonucleotide.
The mixture was extended with Klenow, ligated, and transformed
into BMH 7l-18 mut.9.
After passaging
of phage through BMH 71-18 mut.9, recombinant
phage were selected
on a nonsuppressing
strain, MK30-3.
The
desired phage were identified by screening of RF DNAs for new restriction sites created by silent mutations introduced
by mutagenesis.
Oligonucleotides
used to create As, Ae, A,A&&,,
R,,z -E,R&,
El R2Q9,
and Q are listed in order with the altered bases underlined
(newly
created restriction
sites are indicated):
ACAGCCCAGGTCGCGATATGGTTTAAG
(Nrul)
GATATGGTTTGCGAACCGTCGACGTCGTCAC
(Sall)
CAGCCCAGGTGGCGGCCTGGTTBGAACCGTCGACGTCGTCA
CAAG (Nrul, Sall)
CTCACAGCCCCACGACGTGCGGATCTG
(Dralll)
ATCTGGTTTCAGACCCGTCGACGTCGTCAC
(Bglll. Sall)
ATATGGTTTCAGAACCGTCGACGTCGTCAC
(Sall)
Note that the “template” bicoid single-stranded
DNAs used for mutagenesis with these six oligonucleotides
were wild-type, wild-type, A,,
E1R2Q9, E,Rz, and wild-type, respectively.
Once obtained, the desired
bicoid derivatives
were recloned as EcoRl fragments
into pSH2-1. All
base substitutions
introduced
by these procedures
were confirmed
by
DNA sequence
analysis.
Acknowledgments
We are grateful to Jen Sheen for her expertise on mutagenesis
procedures, Marie-Laure
Samson for plasmids and communication
of unpublished data, and Joanne Kamens and David Eisenmann
for plasmid vectors and helpful advice. We thank Aneel Aggarwal, Thomas
Burglin, David Eisenmann,
Ann Ephrussi, Mike Finney, Erica Golemis,
Gary Ruvkun, and Karen Lech for helpful discussions
and/or comments on the manuscript.
This work was supported by Hoechst AG and
an award to R. B. from the Pew Scholars Program. S. D. H. is a fellow
of the Anna Fuller Foundation.
The costs of publication of this article were defrayed in part by the
payment
of page charges.
This article must therefore
be hereby
marked “advertisement”
in accordance
with 18 U.S.C. Section 1734
solely to indicate this fact.
Received
February
24, 1989; revised
May 23, 1989.
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