Báo cáo khoa học: Site-directed mutagenesis and footprinting analysis of the interaction of the sunflower KNOX protein HAKN1 with DNA ppt

Site-directed mutagenesis and footprinting analysis
of the interaction of the sunflower KNOX protein HAKN1
with DNA
Mariana F. Tioni, Ivana L. Viola, Raquel L. Chan and Daniel H. Gonzalez
Ca
´
tedra de Biologı
´
a Celular y Molecular, Facultad de Bioquı
´
mica y Ciencias Biolo
´
gicas, Universidad Nacional del Litoral, Santa Fe, Argentina
Homeobox genes encode a group of eukaryotic tran-
scription factors generally involved in the regulation of
developmental processes [1]. These genes contain a
region coding for the homeodomain, a 60 amino acid
protein motif that interacts specifically with DNA [2].
The homeodomain folds into a characteristic three-
helix structure. Helices I and II are connected by a
loop, while helices II and III are separated by a turn,
resembling prokaryotic helix-turn-helix transcription
factors. However, unlike helix-turn-helix-containing
proteins, most homeodomains are able to bind DNA
as monomers with high affinity, through interactions
made by helix III (the so-called recognition helix) and
a disordered N-terminal arm located beyond helix I
[3–6].
In plants, the first homeobox was identified in the
maize gene Knotted1 (kn1; [7]). Dominant mutations in
kn1, which is normally active only in meristematic

cells, affect leaf development due to its aberrant
expression in these organs [8]. Additional kn1-like
genes (also termed knox genes) have been isolated
from maize and other monocot and dicot species
Keywords
DNA-binding specificity; footprinting;
homeodomain; KNOX protein; recognition
code
Correspondence
D. H. Gonzalez, Ca
´
tedra de Biologı
´
a Celular
y Molecular, Facultad de Bioquı
´
mica y
Ciencias Biolo
´
gicas (UNL), CC 242 Paraje El
Pozo, 3000 Santa Fe, Argentina
Fax ⁄ Tel: +54 342 4575219
E-mail:
(Received 13 July 2004, revised 31 August
2004, accepted 21 September 2004)
doi:10.1111/j.1432-1033.2004.04402.x
The interaction of the homeodomain of the sunflower KNOX protein
HAKN1 with DNA was studied by site-directed mutagenesis, hydroxyl
radical footprinting and missing nucleoside experiments. Binding of
HAKN1 to different oligonucleotides indicated that HAKN1 prefers the

sequence TGACA (TGTCA), with changes within the GAC core more pro-
foundly affecting the interaction. Footprinting and missing nucleoside
experiments using hydroxyl radical cleavage of DNA showed that HAKN1
interacts with a 6-bp region of the strand carrying the GAC core, covering
the core and nucleotides towards the 3¢ end. On the other strand, protec-
tion was observed along an 8-bp region, comprising two additional nucleo-
tides complementary to those preceding the core. Changes in the residue
present at position 50 produced proteins with different specificities. An
I50S mutant showed a preference for TGACT, while the presence of lysine
shifted the preference to TGACC, suggesting that residue 50 interacts with
nucleotide(s) 3¢ to GAC. Mutation of Lys54 fi Val produced a protein
with reduced affinity and relaxed specificity, able to recognize the sequence
TGAAA, while the conservative change of Arg55 fi Lys completely abol-
ished binding to DNA. Based on these results, we propose a model for the
interaction of HAKN1 with DNA in which helix III of the homeodomain
accommodates along the major groove with Arg55, Asn51, Lys54 and
Ile50, establishing specific contacts with bases of the GACA sequence or
their complements. This model can be extended to other KNOX proteins
given the conservation of these amino acids in all members of the family.
Abbreviations
TALE, three-amino-acid loop extension.
190 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
(reviewed in [9]), indicating that this class of genes
constitutes a family present throughout the plant king-
dom. The knox family of genes can be subdivided into
two classes, I and II, by sequence relatedness and
expression patterns [10]. Based on the expression pat-
terns [11–13], analysis of mutants [14–17] and over-
expression studies [18–21] it was proposed that class
I knox genes are involved in the maintenance of meris-

tematic cells in an undifferentiated state. Indeed, over-
expression of some class I genes in Arabidopsis and
tobacco produces the proliferation of meristems on the
surface of leaves.
The proteins encoded by knox genes belong to the
three-amino-acid loop extension (TALE) superclass.
Members of this superclass contain three extra amino
acids within the loop connecting helices I and II [22]
and are present in several eukaryotic kingdoms, sug-
gesting that they represent an early evolutionary acqui-
sition.
Concerning their interaction with DNA, studies with
proteins from barley [23], tobacco [24], rice [25] and
maize [26] indicate that they bind related sequences
containing a TGAC core (GTCA in the complement-
ary strand), considerably different from the sequence
TAAT recognized by most homeodomains [27]. Eluci-
dating the structural basis for this difference would
help to understand at the molecular level how KNOX
transcription factors recognize their DNA target site.
In this study, we analysed the interaction of the
homeodomain of HAKN1, a sunflower class I KNOX
protein [28], with DNA. Based on studies of wild-type
and mutant forms of the homeodomain, we propose a
model for the complex between HAKN1 and its target
site. This model must be applicable to all KNOX
homeodomains, as important amino acids are con-
served within this family.
Results
Expression and DNA binding analysis of the

HAKN1 homeodomain
The homeodomain of the KNOX transcription factor
HAKN1 was expressed in Escherichia coli as a fusion
with the maltose binding protein using vector
pMALc2. The fusion protein was purified by affinity
chromatography in amylose resin and used for DNA–
protein interaction studies. A 24-bp oligonucleotide
(HAKN1 binding site; BS1) containing the sequence
TGT(G ⁄ C)ACA was used as DNA target. This seq-
uence was designed against a compilation of sequences
bound by KNOX transcription factors from different
species, and contains the TGAC (GTCA) core that is
present in all of them.
Figure 1A shows an electrophoretic mobility shift
assay performed with HAKN1 and oligonucleotide
BS1 or variants containing changes at single positions
(sequences shown in the right panel). We have arbi-
trarily numbered from 1 to 7 those positions present in
the strand that contains the central G. Two shifted
B
A
C
Fig. 1. Binding of HAKN1 to different oligo-
nucleotides. (A) Electrophoretic mobility
shift assay performed with 30 ng of HAKN1
and oligonucleotides containing different
variants of the sequence TGT(G ⁄ C)ACA
(numbers indicated above each lane). (B)
Competition assay of HAKN1 binding to BS1
using a 15-fold molar excess of different

oligonucleotides (numbers indicated above
each lane) as competitors. The sequence of
the 7-bp core present in each oligonucleo-
tide is shown in (C) for reference. Modifica-
tions with respect to BS1 are shown within
black boxes.
M. F. Tioni et al. KNOX homeodomain–DNA interactions
FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 191
bands of similar intensity were observed in this experi-
ment. The relative intensity of the low mobility com-
plex varied when different protein preparations were
used. We speculate that this behavior may arise from
aggregation of the protein. Nevertheless, different pro-
tein preparations showed the same specificity and affin-
ity when considering the amount of bound protein as
the sum of both shifted bands. These bands displayed
similar footprinting patterns (see below), suggesting
that a single HAKN1 homeodomain is bound to DNA
in both complexes. This is strengthened by the fact
that only monomer–DNA complexes were observed in
crosslinking experiments (data not shown).
Analysis of the interaction of HAKN1 with different
oligonucleotides indicates that modifications in the
outermost positions (1 and ⁄ or 7) do not significantly
affect binding (Fig. 1A, lanes BS1, 1,7, 7T, 1 and 7C),
while certain inner nucleotides, notably those located
at positions 4–6, are critical for binding (Fig. 1A, lanes
4, 6A and 5). Regarding position 7, the change of A
for T does not seem to affect binding, while the intro-
duction of C partially reduces the amount of complex

formed. Mutations at positions 2 (not shown) and 3
(lane 3) have only a moderate effect. Similar obser-
vations could be made in experiments in which the
binding to oligonucleotide BS1 was competed with a
15-fold molar excess of different oligonucleotides
(Fig. 1B). These results indicate that HAKN1 mainly
recognizes the GAC (GTC) trinucleotide and displays
lower specificity at outer positions. The GAC triplet is
contained within the TGAC sequence, found to be
part of the binding sites of the barley KNOX protein
Hooded [23] and of maize Knotted1 [26]. This element
is also present in the sequence GTNAC, postulated to
be important for the binding of the tobacco protein
NTH15 to DNA [24], provided that N is G or C.
Analysis of DNA binding by hydroxyl radical
footprinting and interference assays
A more detailed picture of the binding of HAKN1 to
its target site was obtained by the analysis of footprint-
ing patterns after cleavage of free and protein-bound
DNA with hydroxyl radicals generated by Fe–EDTA
complexes. For this purpose, a dimer of the corres-
ponding oligonucleotide ligated through its EcoRI
cohesive site was cloned into the BamHI site of pBlue-
script SK
–
. Cleavage with HindIII and XbaI produces
a 94-bp fragment that contains two HAKN1 binding
sites in opposite orientations. After HAKN1 binding
to the 94-bp oligonucleotide, labeled specifically at one
of its 3¢ ends by filling-in the HindIII site, the complex

was subjected to hydroxyl radical attack, and free and
bound DNA were separated, recovered from the gel
and analysed by denaturing polyacrylamide gel electro-
phoresis (Fig. 2A). Because the oligonucleotide con-
tains two sites in opposite orientation, both strands of
the binding site can be observed in a single footprint-
ing assay. Analysis of the cleavage patterns indicates
that HAKN1 protects six nucleotides from the strand
carrying the sequence TGTGACA (hereafter named
the top strand). The protected area includes GACA
and two adjacent nucleotides (GA) towards the 3¢ end
(Fig. 2A). On the bottom strand, the protected region
covers two additional nucleotides, AC complementary
to GT in TGTGACA (Fig. 2A). For both strands, the
highest protection is observed within the GAC core,
suggesting that the protein makes closer contacts in
this region. This agrees with the important role of
these nucleotide positions in determining the binding
strength of HAKN1 to DNA shown by electrophoretic
mobility shift assays. When the oligonucleotide labeled
at its XbaI site (at the opposite 3¢ end) was used, foot-
printing patterns were identical to those described
above, indicating that HAKN1 makes equivalent
contacts with both binding sites present in the 94-bp
fragment.
Footprinting analysis was also performed with a
similar oligonucleotide containing two mutated sites
[BS(mut1,7);
AGTGACT instead of TGTGACA,
mutations underlined). The results obtained were

essentially the same (not shown), suggesting that
HAKN1 contacts the nucleotide adjacent to the GAC
core and its complement on the other strand whether
they are A or T.
Information about the nucleotide positions that
influence binding of HAKN1 to DNA was obtained
from missing nucleoside (interference) experiments.
Here, DNA is treated with hydroxyl radical-generating
agents before protein binding, thus producing a popu-
lation of molecules with single cleavages along the
phosphodiester backbone. This population is incubated
with the protein of interest and subjected to an elec-
trophoretic mobility shift assay from which the free
and bound fractions are recovered. Molecules with
cleavages at positions important for binding are then
under-represented in the bound fraction and, depend-
ing on the binding conditions, over-represented in the
free fraction. Figure 2B shows a missing nucleoside
experiment using HAKN1 and the 94-bp DNA frag-
ment containing two binding sites previously labeled in
one of its 3¢ ends (HindIII or XbaI sites) and treated
with Fe–EDTA. It is noteworthy that there is a good
correlation between the region protected by HAKN1
and the nucleotide positions important for binding.
This means that all nucleotides in the protected area
KNOX homeodomain–DNA interactions M. F. Tioni et al.
192 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
establish contacts that contribute to binding efficiency.
Again, the GAC core seems to be particularly import-
ant, but outside positions are also required (Fig. 2B).

Within the core, modifications to G and A or their
complements influence binding more markedly. These
results agree with the fact that mutations of these two
nucleotides abolish binding of HAKN1 to DNA. On
the other hand, because nucleotides at outside posi-
tions can be mutated without significant loss in bind-
ing efficiency, it can be assumed that they mainly
participate in nonspecific contacts, such as those estab-
lished with the sugar–phosphate backbone.
The results of footprinting and missing nucleoside
experiments also indicate that HAKN1 does not make
AB
Fig. 2. Hydroxyl radical footprinting and interference analysis of HAKN1 binding to DNA. An oligonucleotide containing two HAKN1 binding
sites (BS1) in opposite orientations was labeled in the 3¢ end of either strand (HindIII or XbaI sites) and subjected to hydroxyl radical attack
either after (A) or before (B) HAKN1 binding. Free (F) and bound (B) DNA were separated and analysed. A portion of the same fragment
digested with defined restriction enzymes was used as a standard (S) to calculate the position of the footprint. Letters to the right of each
panel indicate the DNA sequence (5¢ end in the upper part) of the corresponding strand in this region. In the lower part, the sequence of the
binding site is shown and the protected positions are indicated in bold and underlined. The GAC (GTC) core that shows the highest protec-
tion is shaded.
M. F. Tioni et al. KNOX homeodomain–DNA interactions
FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 193
symmetrical contacts with its target site. The protein
establishes contacts with both strands at the right side
of the GAC core, while only one strand seems to be
contacted at the left side. This lack of symmetry and
the extension of the contacts most probably indicate
that only one molecule of HAKN1 is bound at each
target site.
Binding of HAKN1 single-site mutants to DNA
The picture that emerges from our results is that

HAKN1 binds an 8-bp region of DNA with a tGACa
(tGTCa) specificity core. An interesting question is
how the HAKN1 homeodomain interacts with this
sequence and which amino acids are involved in
sequence-specific contacts. To answer this, we have
analysed the effect of single-site mutations on HAKN1
binding to TGACA and variants of this sequence. It is
logical to assume that changes in amino acids involved
in the interaction must influence binding efficiency. In
addition, some substitutions may alter binding specific-
ity, indicating the existence of contacts between a given
residue and defined positions within the DNA.
Residue 50 (53 in TALE homeodomains) is usually
involved in determining the different specificities
among related homeodomains [27,29–31]. In homeo-
domains that bind the canonical TAAT sequence,
residue 50 interacts with nucleotides located 3¢ to this
site [27,31]. We reasoned, then, that changing Ile50,
present in HAKN1 and all KNOX proteins, may influ-
ence sequence preferences at external positions of the
core. As a first approach, we mutated Ile50 to Ser, pre-
sent in the yeast TALE protein MATa2 [32]. The ana-
lysis of binding of I50S–HAKN1 to variants of the
HAKN1 binding site indicates a preference for an
oligonucleotide containing the sequence TGACT, while
the wild-type HAKN1 homeodomain binds TGACA
and TGACT with similar efficiency (Fig. 3A). This
suggests that residue 50 interacts with the 3¢ region of
the top strand (and ⁄ or the 5¢ region of the bottom
strand), outside the GAC core. This is also evident in

competition experiments (Fig. 3B), where oligonucleo-
tides BS(mut1,7) and BS(mut7T) compete more effi-
ciently than variants with A [BS1 and BS(mut1)] or C
[BS(mut7C)] at this position. Changes at other posi-
tions within the target DNA sequence produced sim-
ilar effects on binding than with the wild-type protein
(Fig. 3).
To further explore the hypothesis that residue 50
is oriented towards the 3¢ end of the top strand, we
also mutated Ile50 to Lys, present in Drosophila bicoid
[33]. I50K–HAKN1 shows a net preference for an
oligonucleotide containing the sequence TGACCC
[BS(mut7C)] over the original TGACAG, present in
AB
CD
Fig. 3. DNA binding preferences of HAKN1
mutants at position 50. (A) Electrophoretic
mobility shift assay of I50S–HAKN1 (30 ng)
binding to BS1 and BS(mut1,7). (B) Binding
of I50S–HAKN1 to BS(mut1,7) was com-
peted with a 100-fold molar excess of oligo-
nucleotides with different sequences
(depicted in Fig. 1). (C) Binding of I50K–
HAKN1 (30 ng) to different oligonucleotides
was analysed by an electrophoretic mobility
shift assay. In (D), the binding of different
amounts (50, 100 and 250 ng) of either
HAKN1 or I50K-HAKN1 to oligonucleotides
BS1 and BS(mut7C) is shown. Oligonucleo-
tide sequences are shown in Fig. 1.

KNOX homeodomain–DNA interactions M. F. Tioni et al.
194 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
BS1 and BS(mut1) (Fig. 3C). This result confirms that
residue 50 interacts with nucleotides adjacent to the
TGAC core. Binding analysis with different oligonuc-
leotides indicated that I50K–HAKN1 is also able to
interact with oligonucleotide BS(mut6G), that contains
a TGAG core (Fig. 3C). In fact, when higher protein
concentrations were used in the assays, binding to
TGAGAG was considerably better than to TGACAG
(not shown), suggesting that Lys50 may also be able to
contact the fourth position of the core, thus changing
the preference for G. The inclusion of Lys at position
50, in addition to promoting a change in specificity,
resulted in a protein with increased affinity towards its
preferred binding site (Fig. 3D). An additional, fast-
migrating band observed in this experiment is present
in free DNA and may represent noncovalent oligo-
nucleotide dimers interacting through their cohesive
ends. We have observed that the presence of this spe-
cies does not affect the intensity of the shifted band.
The increased affinity dispalyed by I50K–HAKN1
may arise from the fact that lysine is able to establish
hydrogen bonds with DNA, which are more stable
than the van der Waals contacts established by Ile.
The interaction of mutants at position 50 with their
preferred binding sites was also analysed by footprint-
ing experiments. I50S–HAKN1 protects a region cov-
ering five nucleotides of the top strand and six
nucleotides of the bottom strand (Fig. 4A). This region

is coincident with the one more strongly protected by
wild-type HAKN1, but is shorter towards the 3¢ end
of the top strand. This result further suggests that
Ile50 contacts the nucleotides located 3¢ to the TGAC
core, as its replacement by a smaller residue such as
Ser allows better access of this region to the modifying
agent. Conversely, I50K–HAKN1 shows an extended
footprinting pattern towards the 3¢ end of the top
strand and the 5¢ end of the bottom strand (Fig. 4B).
This agrees with the presence of a larger residue that
makes stable contacts with this region of DNA.
The interaction of mutants at position 50, and par-
ticularly of I50K–HAKN1 with DNA, provides a
framework to build a model of HAKN1–DNA inter-
actions, taking into account experiments performed with
other homeodomains. The protein bicoid, for example,
is able to bind the sequence TAATCC that contains
the canonical TAAT box [31]. Lys50 of bicoid puta-
tively interacts with the CC dinucleotide, as its muta-
tion to Gln changes its preference to TAATTG [29]. A
reciprocal change, Gln50 to Lys, in engrailed or fushi
tarazu shifts sequence preferences from TAATTA or
TAATTG to TAATCC [30,34]. We postulate, then,
that positioning of the HAKN1 homeodomain along
the TGAC core in DNA must be equivalent to that
adopted by other homeodomains along the TAAT
sequence. The third position of both sequences con-
tains an adenine, known to interact with Asn51,
AB
Fig. 4. Hydroxyl radical footprinting of I50S–HAKN1 (A) and I50K–HAKN1 (B) bound to their preferred binding sites. After binding and hydro-

xyl radical attack, free (F) and bound (B) DNA were separated and analysed. The left and right panels in (A) and (B) represent the top and
bottom strands of the binding site, respectively. A portion of the same fragment digested with defined restriction enzymes was used as a
standard to calculate the position of the footprint. Letters to the right of each panel indicate the DNA sequence (5¢ end in the upper part) of
the corresponding strand in this region. Below the footprints, the sequence of the corresponding binding site is shown and the protected
positions are indicated in bold and underlined.
M. F. Tioni et al. KNOX homeodomain–DNA interactions
FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 195
universally conserved among homeodomains [2,31].
The importance of this interaction is reflected by the
fact that this nucleotide cannot be mutated without a
complete loss of HAKN1 binding. The fourth base in
TAAT is usually recognized by a nonpolar amino acid
(mostly Ile or Val) present at position 47 [2,31].
HAKN1 contains Asn at this position, which may be
too small to establish specific contacts with bases.
Asn47 does not make specific contacts in the homeo-
domain–DNA complexes of MATa2 and extradenticle
[5,35]. Here, we favour the hypothesis that the fourth
position of the core is contacted by Lys54, because the
nucleotide next to that contacted by Asn51 is recog-
nized by residue 54 in other homeodomains (see
below). In support of a prominent role of Lys54, its
mutation to Val produces a significant decrease in
DNA binding (not shown). In addition, K54V–
HAKN1 binds with similar efficiency to sequence vari-
ants containing either A [BS(mut6A)] or C (BS1) at
the fourth position of TGAC, suggesting that it has a
decreased discrimination capacity with respect to wild-
type HAKN1 (Fig. 5). An oligonucleotide containing
TGAG [BS(mut6G)], however, is bound with reduced

efficiency, suggesting that the mutant homeodomain
retains partial specificity. Discrimination at other posi-
tions of the bound region is similar to those displayed
by the wild-type protein. Although the results obtained
do not necessarily indicate a direct role of Lys54 in
establishing contacts with DNA, a plausible explan-
ation is that this residue interacts with at least one of
the members of the CÆG pair at the fourth position of
TGAC in the HAKN1–DNA complex.
The two leftmost positions of the core interact
through the minor groove with the N-terminal arm in
most homeodomains [2,31]. In yeast MATa2, for
example, the N-terminal arm makes base-specific con-
tacts with the first two nucleotides of a TTAC core [5].
Hence, we replaced the N-terminal arm (residues 1–9)
of HAKN1 with the same portion of MATa2, to
determine if a change in specificity was observed. The
resulting protein, Na–HAKN1, showed an overall
reduced affinity but the same sequence preferences as
HAKN1 (Fig. 6). It is noteworthy that it did not bind
oligonucleotide BS(mut4), which contains a TTAC
core on the complementary strand. This indicates that
the N-terminal arm of MATa2 is not able to interact
with DNA within the context of the HAKN1 homeo-
domain as it does within MATa2. Poor binding may
arise from incorrect folding of the chimeric protein or
from the fact that important contacts with DNA are
lost upon replacement of the HAKN1 N-terminal arm.
In addition to a role of the N-terminal arm in contact-
ing the first two amino acids of the core, examination

Fig. 6. Effect of changes within the N-terminal arm and position 55
on the binding of HAKN1 to oligonucleotides BS1 and BS(mut4).
Binding to oligonucleotides containing the sequences TGACA (BS1)
or TTACA [BS(mut4)] was analysed using 30 ng of proteins HAKN1,
R55K–HAKN1, Na–HAKN1 (a protein containing the N-terminal arm
of MATa2) or R55K–Na–HAKN1 (a protein with both modifications).
A
B
Fig. 5. K54V–HAKN1 shows relaxed specificity. Binding of K54V–
HAKN1 (150 ng) to different oligonucleotides was analysed in an
electrophoretic mobility shift assay (A). (B) Competition of K54V–
HAKN1 binding to BS1 with a 25-fold molar excess of different
oligonucleotides (depicted in Fig. 1).
KNOX homeodomain–DNA interactions M. F. Tioni et al.
196 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
of other homeodomain–DNA complexes suggests the
possibility that Arg55 recognizes the second position
of TGAC. Arg55 participates in binding to G residues
in other homeodomains, such as yeast MATa1
(GATG; [36]) or Drosophila extradenticle (TGAT;
[35]). Consistent with a role in DNA binding, an
Arg55 to Ala mutation completely disrupts the inter-
action of HAKN1 with DNA (not shown). To further
analyse its involvement in base-specific contacts, we
reasoned that a conservative substitution for Lys
would not affect nonspecific interactions (i.e. electro-
static interactions with the phosphate backbone), but
would preclude the establishment of hydrogen bonds
with the guanine base of G. The results shown in
Fig. 6 indicate that R55K–HAKN1 is unable to bind

DNA, supporting the hypothesis that Arg55 is
involved in base-specific contacts, which are disrupted
upon mutation to Lys. Another explanation would be
that this change disturbs the overall folding of the
homeodomain, but this seems unlikely because several
homeodomains, notably MATa2, contain Lys at posi-
tion 55. Assuming that the N-terminal arm of MATa2
and Arg55 may be incompatible as both portions may
interact with the same positions of the target site, we
also constructed a mutant in which the N-terminal
arm of MATa2 was inserted into the R55K mutant of
HAKN1. This protein was also ineffective in binding
to the HAKN1 target site or its variants (Fig. 6).
A model for the HAKN1–DNA interaction
Based on the analysis of the binding of wild-type and
mutant HAKN1 homeodomains to different DNA tar-
get sites, we propose a model for the interaction of
HAKN1 with DNA. A set of four amino acids,
located within helix III of the homeodomain, would
make base-specific contacts with defined nucleotides
within the tGACAg sequence. Arg55 would establish a
pair of hydrogen bonds with positions O6 and N7 of
guanine in GACA. As mentioned above, similar inter-
actions have been observed in complexes of other
homeodomains with DNA [35,36]. Asn51 would inter-
act with the first adenine in GACA, also establishing
a pair of hydrogen bonds, as in most homeodomain–
DNA complexes. The next position (C, or G in the
opposite strand) would be contacted by Lys54.
Although there is no evidence in the literature about a

specific contact made by a lysine at this position, resi-
due 54 interacts with the nucleotide adjacent to that
bound by Asn51 in several homeodomains, for exam-
ple MATa2 (Arg54, TTAC; [5]), TTF1 (Tyr54, CAAG;
[37]), bicoid (Arg54, TAAT or TAAG; [38]) and Hahr1
(Thr54, TAAA, in this case in combination with
Phe47; [39]). Additionally, lysine determines a prefer-
ence for C at an adjacent position when present at
position 50 in bicoid and other mutant homeodomains
(including HAKN1, see above), presumably by inter-
acting with guanine bases through hydrogen bonds as
observed in the Lys50–engrailed crystal structure [40].
Finally, our results also indicate that Ile50 is
involved in establishing a preference for A or T at the
3¢ side of the core. Mutations of this residue to Ser or
Lys were able to confer a new binding specificity to
HAKN1, changing to a net preference for T or C,
respectively. Ile50 is present in MATa1, where it inter-
acts with a TA dinucleotide adjacent to the position
contacted by Met54 [36]. Accordingly, Ile50 may also
be involved in contacts with an adjacent position,
which is protected by HAKN1 in footprinting experi-
ments and interferes with binding when modified by
hydroxyl radical attack.
To examine the consistence of the interactions des-
cribed above, we have constructed a theoretical model
of the HAKN1–DNA complex using the program
swiss-model [41] available in the ExPASy web server.
Different models for wild-type and mutant HAKN1
were obtained using the homeodomain–DNA com-

plexes of extradenticle [35], MATa1 [36] and MATa2
[5] as templates. Figure 7 shows the alignment of helix
III of the HAKN1 homeodomain along the major
groove of DNA (the MATa1 binding site in this case).
Amino acids in red are those present in wild-type
HAKN1 that putatively contact the GAC core. Note
that Arg55 and Asn51 establish hydrogen bonds with
adjacent G and A, respectively. Interestingly, Lys54
also appears making a hydrogen bond with the N7 of
an adjacent purine (adenine in this case) present in the
complementary strand. A similar contact could be
made with a guanine complementary to C in GAC,
further suggesting that Lys54 is likely to contact this
position. The position of Lys55 in the corresponding
mutant is shown in yellow. Clearly, the specific con-
tacts made by Arg55 are lost and are replaced by an
interaction with the phosphate backbone. Val54 is
shown in pink. The shorter side chain and the loss of
a hydrogen bond explain the decrease in affinity and
relaxed specificity. Finally, the variants at position 50
(Ile in orange, Ser in green and Lys in blue) are also
represented. All these residues are oriented towards the
3¢ end of the core and probably establish contacts with
the complementary strand. It should be emphasized
that the mutagenesis experiments described here do
not prove that certain amino acids make base-specific
contacts, especially when a new specificity at a defined
position was not achieved. The combination of these
experiments with the footprinting results and previous
M. F. Tioni et al. KNOX homeodomain–DNA interactions

FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 197
knowledge, however, are highly indicative that this is
the case. Determination of the three-dimensional struc-
ture of the complex will be required to evaluate the
accuracy of the DNA–protein contacts proposed by
this model.
Discussion
In this study, we investigated the interaction of the
homeodomain of the KNOX protein HAKN1 with
DNA. As no structural studies on the interaction of
any KNOX protein with DNA have been reported,
ours constitutes a first approach to understand these
interactions at the molecular level. Electrophoretic
mobility shift assays, footprinting analyses and missing
nucleoside experiments using different binding sites
and mutated proteins allowed us to establish a model
for HAKN1–DNA interaction. This model postulates
that HAKN1 binds to a TGACNN core primarily
through interactions of certain helix III amino acids
(Ile50, Asn51, Lys54 and Arg55) with DNA. This
particular combination of amino acids is present only
in KNOX proteins, indicating that they may have been
selected through evolution to generate a defined specif-
icity. Among them, the incorporation of Ile50 and
Arg55 must have been particularly important. Other
homeodomains that contain Ile50 and Arg55 are those
of the TGIF, Meis and Bell families [22] and yeast
MATa1 [36]. TGIF and Meis proteins bind the
sequence TGTCA (TGACA on the complementary
strand [42,43]), which is identical to that recognized by

HAKN1. They possess Arg at position 54, suggesting
that Lys54 in HAKN1 may not be the only means of
recognizing the TGAC core. Accordingly, the Bell
protein ATH1, which contains Val54, also selects
the sequence TGACA from a random population
(I. Viola, unpublished results). The presence of Val54
produces, however, a relaxed specificity at the fourth
position and reduced affinity within the context of
both the HAKN1 (this study) and the ATH1 homeo-
domain (I. Viola, unpublished results). MATa1, in
turn, binds a completely different sequence (GATGT ⁄
ACATC [44]), indicating that other factors apart from
these residues also influence specificity. The GA dinu-
cleotide in GATGT is recognized by Arg55 and Asn51
of MATa1, as proposed here for the GA dinucleotide
in TGAC. The GT dinucleotide is, in turn, contacted
by Met54 and Ile50 through interactions with the com-
plementary strand [36]. This means that, in MATa1,
positions contacted by residues 55 ⁄ 51 and 54 ⁄ 50 are
separated by one additional base pair. This may be
originated by the presence of Val47, which binds the
nucleotide adjacent to the A recognized by Asn51 in
many homeodomains. The above-mentioned TGAC
binding proteins (including KNOX proteins) contain
Asn47, which may not establish specific contacts with
DNA. These differences may also originate changes in
the relative orientation of DNA contacting amino
acids.
The model presented here can also be compared
with the structures determined for the TALE proteins

extradenticle and PBX1 bound to DNA [35,45]. These
proteins bind the sequence TGAT, with Arg55 and
Asn51 establishing hydrogen bonds with the GA dinu-
cleotide, as proposed here for HAKN1. The initial T is
also contacted by Arg55 through van der Waals inter-
actions in the PBX1 complex [45]. The second T makes
van der Waals contacts with Asn47. As PBX1 and
extradenticle contain Ile54, this situation may resemble
the binding behaviour of K54V–HAKN1, which shows
relaxed specificity at this position.
Our results with HAKN1 clearly support the idea
that there is a general recognition code for homeo-
domains. Accordingly, recognition at the left side of
A
B
Fig. 7. A model for the interaction of HAKN1 with DNA. (A) Dia-
gram of the HAKN1 DNA binding site with the residues putatively
involved in binding each position. (B) Spatial model of the interac-
tion of helix III of wild-type and mutant HAKN1 homeodomains
with DNA. The model was constructed with the program
SWISS-
MODEL [41] using the structures of the DNA complexes of MATa1
(1YRN), extradenticle (1B8I) and MATa2 (1APL) as templates.
Amino acids in red are those present in wild-type HAKN1 that puta-
tively contact the GAC core. Residues at position 50 are: Ile in
orange, Ser in green and Lys in blue. Val54 and Lys55, present in
the mutants, are shown in pink and yellow, respectively.
KNOX homeodomain–DNA interactions M. F. Tioni et al.
198 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
the conserved A that is contacted by the universally

present Asn51 is determined by the N-terminal arm
and ⁄ or Arg55. The presence of Arg55 determines a G
5¢ to the conserved A, while the N-terminal arm seems
to determine a preference for A ⁄ T base pairs. The set
of residues present at positions 47, 50 and 54 influence
binding preferences at the right side.
The putative DNA-contacting amino acids of
HAKN1 are also present in all described KNOX pro-
teins, indicating that they may all recognize identical
or similar sequences. This raises the question of how
the specificity of interaction is achieved in vivo, because
different KNOX proteins have different functions.
A similar paradox has been noted for animal homeo-
domains, for which current evidence suggests that spe-
cificity arises from the interaction of homeodomain
proteins with other factors that somehow influence
their DNA binding properties [46]. Plant KNOX pro-
teins interact with proteins from the Bell family, which
also belong to the TALE superclass [47,48] and bind
similar sequences [49]. Chen et al. [49] have shown that
potato KNOX and Bell proteins bind two tandem cop-
ies of a TGAC motif separated by one additional nuc-
leotide. As both types of homedomains seem to
establish similar contacts with their target sites
(I. Viola, unpublished results), this indicates that the
respective recognition helices must lie in an antiparallel
orientation within the major groove at opposite sides
of the DNA. According to the footprinting data pre-
sented here, the central nucleotide pair and the first
two pairs of the second TGAC would be contacted by

both proteins. This may indicate that some rearrange-
ments may occur upon complex formation by KNOX
and Bell proteins, either before or after binding to
DNA. In the complexes formed by PBX1 and extra-
denticle, the presence of Gly50, which does not contact
DNA, may allow the binding of an additional homeo-
domain in tandem immediately following TGAT
[35,43,45]. The presence of Ile50, that interacts with
nucleotides located at the 3¢ side of the core, may
explain the requirement of a larger distance between
both binding sites in the complexes formed by KNOX
and Bell proteins.
Sequences outside the homeodomain may also influ-
ence the binding properties of the protein. Indeed, a
stretch of 16 amino acids located immediately C-ter-
minal to the homeodomain forms an a-helix that has
been shown to influence the DNA binding affinity of
the PBX1 homeodomain [50,51]. As the protein used
in our assays includes a C-terminal portion, we have
analysed the structure of the region immediately fol-
lowing the HAKN1 homeodomain using several secon-
dary structure prediction programs. We have only
observed a short region (five to eight amino acids
depending on the program) that has a propensity to
form an a-helix. Therefore, we consider it unlikely that
an effect of the C-terminal tail, similar to that
observed with PBX1, occurs in HAKN1 or other
KNOX proteins.
In summary, the results presented here constitute a
framework to understand at the molecular level how

KNOX proteins interact with DNA and how these
interactions contribute to the establishment of active
transcription complexes that influence defined develop-
mental pathways within plant cells.
Experimental procedures
Cloning, expression and purification of
recombinant proteins
HAKN1 homeodomain and C-terminal sequences were
amplified and cloned in-frame into the EcoRI and PstI sites
of the expression vector pMAL-c2 (New England Biolabs,
Beverly, MA, USA). Amplifications were performed using
Pfu DNA polymerase and oligonucleotides MALN1: 5¢-GC
GGAATTCAAAAAGAGAAAGAAAGGG-3¢ and MALC:
5¢-GGCCTGCAGCTAGAGAAGTGAAACATC-3¢ with
HAKN1 cDNA [28] as the template.
An I50S mutant was constructed using complementary
oligonucleotides I50SF and I50SR (5¢-CAACTGGTTC
A
GCAACCAAAGGAA-3¢ and 5¢-TTCCTTTGGTTGC
TGAACCAGTTG-3¢; introduced mutations underlined)
together with primers MALC and MALN1, respectively, to
amplify partially overlapping N-terminal and C-terminal
HAKN1 fragments. The resulting products were mixed in
buffer containing 50 mm Tris ⁄ HCl (pH 7.2), 10 mm
MgSO
4
, and 0.1 mm dithiothreitol, incubated at 95 °C dur-
ing 5 min, and annealed by allowing the solution to cool to
24 °C in approximately 1 h. After this, 0.5 mm of each
dNTP and 5 units of the Klenow fragment of E. coli DNA

polymerase I were added, and incubation was followed for
1 h at 37 ° C. An aliquot of this reaction was used directly
to amplify the annealed fragments using primers MALN1
and MALC. Mutants I50K, K54V, R55A and R55K were
constructed in a similar way, using oligonucleotides I50KF
(5¢-CAACTGGTTCA
AAAACCAAAGGAA-3¢), I50KR
(5¢-TTCCTTTGGTTT
TTGAACCAGTTG-3¢), K54VF (5¢-
TAAACCARAGG
GTGCGGCAYTGGA-3¢), K54VR (5¢-
TCCARTGCCGC
ACCCTYTGGTTTA-3¢), R55AF (5¢-CA
AAGGAAG
GCGCACTGGAA-3¢), R55AR (5¢-TTCCAG
TGC
GCCTTCCTTTG-3¢), R55KF (5¢-CAAAGGAAGAA
GCACTGGAA-3¢) and R55KR (5¢-TTCCAGTGC
TTCT
TCCTTTG-3¢) to introduce the mutations. The N-terminal
arm of the MATa2 homeodomain (amino acids 1–9) was
introduced into the HAKN1 homeodomain using two
successive rounds of amplification with oligonucleotides
M. F. Tioni et al. KNOX homeodomain–DNA interactions
FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 199
MAT1 (5¢-AGGGGACATAGATTTACAAAAGAAGCTC
GTCAACAA-3¢; first round, MATa2 sequences underlined)
or MAT2 (5¢-CGCGAATTC
AAGCCGTACAGGGGAC
ATAGATTTACA-3¢; second round) together with oligo-

nucleotide MALC (both rounds). All constructions were
checked by DNA sequence analysis. Expression and purifi-
cation of the recombinant proteins were carried out as indi-
cated by the manufacturers of the pMAL-c2 system. The
amount of purified protein (> 95% as judged by
SDS ⁄ PAGE) was determined as described by Sedmak and
Grossberg [52].
DNA binding assays
For electrophoretic mobility shift assays, aliquots of puri-
fied proteins were incubated with double stranded DNA
(0.3–0.6 ng, 10 000 c.p.m., labeled with [
32
P]dATP[aP] by
filling-in the 3¢-ends using the Klenow fragment of DNA
polymerase) generated by hybridization of the complement-
ary oligonucleotides 5¢-AATTCAGATCTTGTGACAGAA
GAG-3¢ and 5¢-GGATCCTCTTCTGTCACAAGATCTG-
3¢, or derivatives with modifications as described in the text.
Binding reactions (20 lL) containing 20 mm Hepes
(pH 7.5), 50 mm KCl, 2 mm MgCl
2
, 0.5 mm EDTA,
1.0 mm dithiothreitol, 0.5% (v ⁄ v) Triton X-100, 10% (v ⁄ v)
glycerol and 22 ngÆlL
)1
BSA, were incubated for 30 min at
20 °C, supplemented with 2.5% (w ⁄ v) Ficoll and immedi-
ately loaded on to a running gel [5% (w ⁄ v) acrylamide,
0.08% (w ⁄ v) bis-acrylamide in 0.5· TBE plus 2.5% (v ⁄ v)
glycerol; 1· TBE is 90 mm Tris ⁄ borate, pH 8.3, 2 mm

EDTA). The gel was run in 0.5· TBE at 30 mA for 1.5 h
and dried prior to autoradiography.
Footprinting analysis
For the analysis of hydroxyl radical footprinting patterns, a
double-stranded oligonucleotide containing the HAKN1
binding site (BS1) with BamHI and Eco RI compatible
cohesive ends was self-ligated through its EcoRI site to
obtain a dimer and then cloned into the BamHI site of
pBluescript SK
–
. From this clone, a 94-bp fragment con-
taining two HAKN1 binding sites in opposite orientations
was obtained and labeled at one of its 3¢ ends. This was
accomplished by PCR using reverse and universal primers,
followed by cleavage with either HindIII or XbaI (from the
pBluescript polylinker), incubation with the Klenow frag-
ment of DNA polymerase and [
32
P]dATP[aP], cleavage
with the other enzyme and purification by nondenaturing
polyacrylamide gel electrophoresis. Binding of HAKN1 to
this oligonucleotide (200 000 c.p.m.) was performed at
20 °Cin15lLof50mm Tris ⁄ HCl (pH 7.5), 100 mm
NaCl, 10 mm 2-mercaptoethanol, 0.1 mm EDTA,
22 ngÆlL
)1
BSA, 10 ngÆlL
)1
poly(dI-dC) and 800 ng
HAKN1. After 30 min, the binding reaction was subjected

to hydroxyl radical cleavage by the addition of 10.5 lLof
6.6 mm sodium ascorbate, 0.66 mm EDTA (pH 8.0),
0.33 mm (NH
4
)
2
Fe(SO
4
)
2
and 0.2% H
2
O
2
, and the bound
and free oligonucleotides were separated in a nondenatur-
ing gel [53]. The corresponding bands were excised from
the gel, eluted and analysed on denaturing polyacrylamide
gels. To determine the position of the footprint within the
sequence, a portion of the same fragment was digested with
defined restriction enzymes and loaded in the same gel. A
DNA fragment containing two copies of oligonucleotide
BS(mut1,7) was obtained and analysed in a similar way. A
single copy of oligonucleotide BS(mut7C) was cloned into
the BamHI and EcoRI sites of pBluescript SK
–
and used
for footprinting assays as described above for BS1.
Missing nucleoside experiments
For missing nucleoside experiments, one-end-labeled oligo-

nucleotides containing the different binding sites were
obtained from clones in pBluescript SK
–
as described above
and subjected to hydroxyl radical cleavage [53]. Binding of
HAKN1 to the treated oligonucleotide (200 000 c.p.m.) and
separation of the free and bound fractions by electropho-
retic mobility shift assays were performed as described in
the last section. These fractions were excised from the gel,
eluted and analysed on a denaturing polyacrylamide gel as
described above.
Acknowledgements
This work was supported by grants from CONICET,
ANPCyT and Universidad Nacional del Litoral (Argen-
tina). R.L.C. and D.H.G. are members of CONICET;
M.F.T. and I.L.V. are fellows of CONICET.
References
1 Gehring WJ (1987) Homeo boxes in the study of devel-
opment. Science 236, 1245–1252.
2 Gehring WJ, Affolter M & Bu
¨
rglin T (1994) Homeo-
domain proteins. Annu Rev Biochem 63, 487–526.
3 Kissinger CR, Liu B, Martin-Blanco E, Kornberg TB &
Pabo CO (1990) Crystal structure of an engrailed homeo-
domain-DNA complex at 2.8 A resolution: a framework
for understanding homeodomain-DNA interactions. Cell
63, 579–590.
4 Otting G, Qian YQ, Billeter M, Mu
¨

ller M, Affolter M,
Gehring WJ & Wu
¨
thrich K (1990) Protein-DNA con-
tacts in the structure of a homeodomain-DNA complex
determined by nuclear magnetic resonance spectroscopy
in solution. EMBO J 9, 3085–3092.
5 Wolberger C, Vershon AK, Liu B, Johnson AD & Pabo
CO (1991) Crystal structure of a MATa2 homeodo-
main-operator complex suggests a general model for
homeodomain–DNA interactions. Cell 67 , 517–528.
KNOX homeodomain–DNA interactions M. F. Tioni et al.
200 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS
6 Gehring WJ, Qian YQ, Billeter M, Furukubo-Tokunaga
K, Schier AF, Resendez-Perez D, Affolter M, Otting G
&Wu
¨
thrich K (1994) Homeodomain-DNA recognition.
Cell 78, 211–223.
7 Vollbrecht E, Veit B, Sinha N & Hake S (1991) The
developmental gene Knotted-1 is a member of a maize
homeobox gene family. Nature 350, 241–243.
8 Smith LG, Greene B, Veit B & Hake S (1992) A domi-
nant mutation in the maize homeobox gene, Knotted-1,
causes its ectopic expression in leaf cells with altered
fates. Development 116, 21–30.
9 Chan RL, Gago GM, Palena CM & Gonzalez DH
(1998) Homeoboxes in plant development. Biochim
Biophys Acta 1442, 1–19.
10 Kerstetter R, Vollbrecht E, Lowe B, Veit B, Yamaguchi

J & Hake S (1994) Sequence analysis and expression
patterns divide the maize knotted1-like homeobox genes
into two classes. Plant Cell 6, 1877–1887.
11 Jackson D, Veit B & Hake S (1994) Expression of maize
KNOTTED1 related homeobox genes in the shoot api-
cal meristem predicts patterns of morphogenesis in the
vegetative shoot. Development 120, 405–413.
12 Lincoln C, Long J, Yamaguchi J, Serikawa K & Hake
S (1994) A knotted1-like homeobox gene in Arabidopsis
is expressed in the vegetative meristem and dramatically
alters leaf morphology when overexpressed in transgenic
plants. Plant Cell 6, 1859–1876.
13 Sentoku N, Sato Y, Kurata N, Ito Y, Kitano H & Mat-
suoka M (1999) Regional expression of the rice KN1-
type homeobox gene family during embryo, shoot, and
flower development. Plant Cell 11, 1651–1663.
14 Mu
¨
ller KJ, Romano N, Gerstner O, Garcia-Maroto F,
Pozzi C, Salamini F & Rohde W (1995) The barley
Hooded mutation caused by a duplication in a homeo-
box gene intron. Nature 374, 727–730.
15 Schneeberger RG, Becraft PW, Hake S & Freeling M
(1995) Ectopic expression of the knox homeobox gene
rough sheath1 alters cell fate in the maize leaf. Genes
Dev 9, 2292–2301.
16 Long JA, Moan EI, Medford JI & Barton MK (1996)
A member of the knotted class of homeodomain pro-
teins encoded by the STM gene of Arabidopsis. Nature
378, 66–69.

17 Kerstetter RA, Laudencia-Chingcuanco D, Smith LG &
Hake S (1997) Loss-of-function mutations in the maize
homeobox gene, knotted1 , are defective in shoot meris-
tem maintenance. Development 124, 3045–3054.
18 Matsuoka M, Ichikawa H, Saito A, Tada Y, Fujimura
T & Kano-Murakami Y (1993) Expression of a rice
homeobox gene causes altered morphology of transgenic
plants. Plant Cell 5, 1039–1048.
19 Sinha NR, Williams EE Jr & Hake S (1993) Over-
expression of the maize homeo box gene, KNOTTED-1,
causes a switch from determinate to indeterminate cell
fates. Genes Dev 7, 787–795.
20 Chuck G, Lincoln C & Hake S (1996) KNAT1 induces
lobed leaves with ectopic meristems when overexpressed
in Arabidopsis. Plant Cell 8, 1277–1289.
21 Tamaoki M, Kusaba S, Kano-Murakami Y & Mat-
suoka M (1997) Ectopic expression of a tobacco homeo-
box gene, NTH15, dramatically alters leaf morphology
and hormone levels in transgenic tobacco. Plant Cell
Physiol 38, 917–927.
22 Bu
¨
rglin TR (1997) Analysis of TALE superclass homeo-
box genes (MEIS, PBC, KNOX, Iroquois, TGIF)
reveals a novel domain conserved between plants and
animals. Nucleic Acids Res 25, 4173–4180.
23 Krusell L, Rasmussen I & Gausing K (1997) DNA
binding sites recognised in vitro by a knotted class I
homeodomain protein encoded by the hooded gene, k,
in barley (Hordeum vulgare). FEBS Lett 408, 25–29.

24 Sakamoto T, Kamiya N, Ueguchi-Tanaka M, Iwahori S
& Matsuoka M (2001) KNOX homeodomain protein
directly suppresses the expression of a gibberellin bio-
synthetic gene in the tobacco shoot apical meristem.
Genes Dev 15, 581–590.
25 Nagasaki H, Sakamoto T, Sato Y & Matsuoka M (2001)
Functional analysis of the conserved domains of a rice
KNOX homeodomain protein, OSH15. Plant Cell 13,
2085–2098.
26 Smith HMS, Boschke I & Hake S (2002) Selective inter-
action of plant homeodomain proteins mediates high
DNA-binding affinity. Proc Natl Acad Sci USA 99,
9579–9584.
27 Laughon A (1991) DNA binding specificity of homeo-
domains. Biochemistry 30, 11357–11367.
28 Tioni MF, Gonzalez DH & Chan RL (2003) Knotted1-
like genes are strongly expressed in differentiated cell
types in sunflower. J Exp Bot 54, 681–690.
29 Hanes SD & Brent R. (1989) DNA specificity of the
bicoid activator protein is determined by homeodomain
recognition helix residue 9. Cell 57, 1275–1283.
30 Ades SE & Sauer RT (1994) Differential DNA binding
specificity of the Engrailed homeodomain: the role of
residue 50. Biochemistry 33, 9187–9194.
31 Wilson DS, Sheng G, Jun S & Desplan C (1996) Con-
servation and diversification in homeodomain–DNA
interactions: a comparative genetic analysis. Proc Natl
Acad Sci USA 93, 6886–6891.
32 Johnson AD & HerskowitZ I (1985) A repressor and its
operator control expression of a set of cell type specific

genes in yeast. Cell 42, 237–247.
33 Berleth T, Burri M, Thoma G, Bopp D, Richstein S,
Frigerio G, Noll M & Nu
¨
sslein-Volhard C (1988) The
role of localization of bicoid RNA in organizing the
anterior pattern of the Drosophila embryo. EMBO J 7,
1749–1756.
34 Percival-Smith A, Mu
¨
ller M, Affolter M & Gehring WJ
(1990) The interaction with DNA of wild-type and mutant
fushi tarazu homeodomains. EMBO J 9, 3967–3974.
M. F. Tioni et al. KNOX homeodomain–DNA interactions
FEBS Journal 272 (2005) 190–202 ª 2004 FEBS 201
35 Passner JM, Ryoo HD, Shen L, Mann RS & Aggarwal
AK (1999) Structure of a DNA-bound Ultrabithorax-
Extradenticle homeodomain complex. Nature 397, 714–
719.
36 Li T, Stark MR, Johnson AD & Wolberger C (1995)
Crystal structure of the MATa1 ⁄ MATa2 homeodomain
heterodimer bound to DNA. Science 270, 262–269.
37 Damante G, Pellizzari L, Esposito G, Fogolari F,
Viglino P, Fabbro D, Tell G, Formisano S & Di Lauro
R. (1996) A molecular code dictates sequence-specific
DNA recognition by homeodomains. EMBO J 15,
4992–5000.
38 Dave V, Zhao C, Yang F, Tung CS & Ma J (2000)
Reprogrammable recognition codes in bicoid homeodo-
main–DNA interaction. Mol Cell Biol 20, 7673–7684.

39 Tron AE, Bertoncini CW, Palena CM, Chan RL &
Gonzalez DH (2001) Combinatorial interactions of two
amino acids with a single base pair define target site
specificity in plant dimeric homeodomain proteins.
Nucleic Acids Res 29, 4866–4872.
40 Tucker-Kellogg L, Rould MA, Chambers KA, Ades
SE, Sauer RT & Pabo CO (1997) Engrailed
(Gln50 fi Lys) homeodomain-DNA complex at 1.9 A
˚
resolution: structural basis for enhanced affinity and
altered specificity. Structure 5, 1047–1054.
41 Schwede T, Kopp J, Guex N & Peitsch MC (2003)
SWISS-MODEL: an automated protein homology-mod-
eling server. Nucleic Acids Res 31, 3381–3385.
42 Bertolino E, Reimund B, Wildt-Perinic D & Clerc RG
(1995) A novel homeobox protein whcih recognizes a
TGT core and functionally interferes with a retinoid-
responsive motif. J Biol Chem 270, 31178–31188.
43 Knoepfler PS, Calvo KR, Chen H, Antonarakis SE &
Kamps MP (1997) Meis1 and pKnox1 bind DNA coop-
eratively with Pbx1 utilizing an interaction surface dis-
rupted in oncoprotein E2a-Pbx1. Proc Natl Acad Sci
USA 94, 14553–14558.
44 Goutte C & Johnson AD (1994) Recognition of a DNA
operator by a dimer composed of two different homeo-
domain proteins. EMBO J 13 , 1434–1442.
45 LaRonde-LeBlanc NA & Wolberger C (2003) Structure
of HoxA9 and Pbx1 bound to DNA: Hox hexapeptide
and DNA recognition anterior to posterior. Genes Dev
17, 2060–2072.

46 Chariot A, Gielen J, Merville MP & Bours V (1999)
The homeodomain-containing proteins: an update on
their interacting partners. Biochem Pharmacol 58, 1851–
1857.
47 Bellaoui M, Pidkowich MS, Samach A, Kushalappa K,
Kohalmi SE, Modrusan Z, Crosby WL & Haughn GW
(2001) The Arabidopsis BELL1 and KNOX TALE
homeodomain proteins interact through a domain con-
served between plants and animals. Plant Cell 13, 2455–
2470.
48 Mu
¨
ller J, Wang Y, Franzen R., Santi L, Salamini F &
Rohde W (2001) In vitro interactions between barley
TALE homeodomain proteins suggest a role for pro-
tein–protein associations in the regulation of Knox gene
function. Plant J 27, 13–23.
49 Chen H, Banerjee AK & Hannapel DJ (2004) The tan-
dem complex of BEL and KNOX partners is required
for transcriptional repression of Ga20ox1. Plant J 38 ,
276–284.
50 Lu Q & Kamps MP (1996) Structural determinants
within Pbx1 that mediate cooperative DNA binding
with pentapeptide-containing Hox proteins: proposal for
a model of a Pbx1-Hox-DNA complex. Mol Cell Biol
16, 1632–1640.
51 Green NC, Rambaldi I, Teakles J & Featherstone MS
(1998) A conserved C-terminal domain in PBX increases
DNA binding by the PBX homeodomain and is not a
primary site of contact for the YPWM motif of

HOXA1. J Biol Chem 273, 13273–13279.
52 Sedmak J & Grossberg S (1977) A rapid, sensitive, and
versatile assay for protein using Coomassie brilliant blue
G-250. Anal Biochem 79, 544–552.
53 Dixon WJ, Hayes JJ, Levin JR, Weidner MF, Dombro-
ski BA & Tullius TD (1991) Hydroxyl radical footprint-
ing. Methods Enzymol 208, 380–413.
KNOX homeodomain–DNA interactions M. F. Tioni et al.
202 FEBS Journal 272 (2005) 190–202 ª 2004 FEBS