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INTRODUCTION |
Hox gene transcription factors are key players in the
establishment of the anteroposterior positional values along the body axis, as shown by the analysis of their mutant phenotypes in animals as
evolutionary diverged as nematodes, insects, and mammals (1). Hox genes are expressed in segmental structures along the
anteroposterior axis in developing vertebrate embryos according to
spatially and temporally restricted patterns, which correlate with
their physical arrangement in the cluster, following the
3'-anterior/early 5'-posterior/late colinearity rule (1-5). The
homeodomain is the DNA-binding motif necessary for the biological
function of HOX proteins. However, despite markedly divergent
biological effects occurring in vivo, the homeodomains of
many different HOX proteins share a very high sequence similarity and
can bind in vitro with similar affinities to many DNA
sites containing an ATTA core sequence (6-8).
Hox genes are expressed in many parts of the vertebrate
central nervous system but are not expressed in the rostral brain, i.e. in forebrain and in midbrain. The early organization of
the developing rostral brain in higher vertebrates and the existence of
a segmental patterning in this region is still the object of intense
debate. The expression pattern of putative regulatory genes has
revealed that the rostral brain can be divided into discrete
longitudinal and transverse domains (9). One of the genes showing such
a regionally restricted expression in the forebrain is Otx2,
a homeobox-containing gene isolated in mouse and humans as one of the
two counterparts of the Drosophila orthodenticle gene, which
is involved in the control of the head development in flies (10-12).
Orthologues of Otx2 have subsequently been cloned in several
other species (13-16). Recently, three groups have shown that targeted
Otx2 inactivation in mice leads to embryonal lethality, with
embryos showing severe gastrulation defects and deletions of rostral
brain (17-19).
Otx2 mRNA and protein are present at 5.5-5.7 days
postcoitum (d.p.c.)1 in the
embryonic portion of the primary ectoderm (or epiblast). Subsequently,
between 7.0 and 7.5 d.p.c., Otx2 expression becomes progressively restricted to the anterior portion of the embryo, mainly
the neuroectoderm of the headfold, fated to give rise to fore- and
midbrain (11, 20). As Otx2 expression turns off in the
posterior regions, Hox genes are progressively activated, in
such a way that Otx2 is eventually restricted to the most
anterior third of the embryo, and Hox genes are restricted
to the most posterior two-thirds (21, 22).
The complex spatial and temporal regulation of the Otx2 and
Hox genes can be mimicked experimentally both in
vivo and in vitro. In fact, during early mouse
development, high doses of retinoic acid (RA) (a molecule involved in
embryonic pattern formation and specification of regional identities in
central nervous system, axial skeleton, and limbs) severely repress
Otx2 expression, leading to a reduction in the extent of
fore- and midbrain at later stages and, conversely, to an expansion of
the hindbrain together with a strong induction of the expression of
Hoxb-1 and Hoxb-2 anteriorly (23, 24). In a human
embryonal carcinoma cell line (NT2/D1), RA treatment abolishes
constitutive Otx2 expression and strongly activates
Hox gene expression (11, 25, 26). In addition, in NT2/D1
cells the activity of a 5' genomic fragment of the mouse Otx2 gene driving a reporter gene showed a 6-9-fold
decrease after RA treatment (23), while the activity of Hox
gene regulatory sequences is strongly enhanced (27-29). Taken
together, these data suggest that the complementary spatial and
temporal regulation of the Otx2 and Hox genes
might be a consequence of the RA gradient present in the embryo and
raise the possibility that Otx2 and Hox gene
expression might mutually exclude each other by means of regulatory
interactions.
The restriction of the Otx2 expression to the anterior
neuroectoderm has been shown to require both positive and negative signals coming from the underlying mesendoderm (21). In this report, we
explore the possibility that anteriorly expressed Hox genes
play a role in the early regulation of Otx2 expression
through activation or repression of the transcriptional activity of the 5' Otx2 genomic sequences. We show that a 1821-bp 5' genomic
fragment of the mouse Otx2 gene, whose activity has already
been shown to be down-regulated by RA (23), can also directly interact with Hox genes. The transcriptional activity of the
5'-flanking Otx2 genomic sequences driving a reporter gene
in NT2/D1 cells was increased by co-transfection with HOXB1, HOXB2, and
HOXB3-encoding expression vectors, suggesting that Hox genes
are not involved in the down-regulation of Otx2 expression
in the posterior part of the embryo, but rather they might be involved
in the refinement of Otx2 expression boundaries in the
anterior compartment. Hox genes expressed more posteriorly
during later stages of development were unable to transactivate
Otx2 expression. The binding sites of the three HOX proteins
have been identified and shown to be necessary for the transactivation
in transfected cells by HOXB1 and HOXB3 but not by HOXB2. The same
sites were also recognized by nuclear extracts obtained from
12.5-day-old mouse embryos. Our data suggest that HOXB1, HOXB2, and
HOXB3 might directly activate the Otx2 promoter and,
possibly, play a role in the early regulation of the Otx2
gene expression.
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EXPERIMENTAL PROCEDURES |
Reporter Plasmid and cDNA Expression Vectors--
To obtain
the pOTX2Luc
1219, the pOTX2Luc
710, and pOTX2Luc
+163
constructs, a 1821-bp EcoRI-StyI, a 1310-bp
ClaI-StyI, and a 439-bp
HaeIII-StyI DNA fragment, respectively, of the
mouse OTX2 genomic upstream sequence, extending to position +600 in their 3'-ends, was cloned into the SmaI site of the pXP2Luc
reporter plasmid (30). The pOTX2LucATTAbox-TK construct was obtained cloning a HinfI-HaeIII 534-bp fragment into the
SmaI site of the pT81Luc reporter plasmid. The HBE1 binding
site was mutagenized by polymerase chain reaction with the mismatched
primers HBE1m (5'-ATCCAGACTACTGCAGAGGTGAAAATGAT-3'), containing a
PstI site in the mutated sequence, to obtain the
pOTX2LucATTAbox-TKm construct.
The pSG-HOXB1 cDNA construct was reconstructed by a 5' genomic
370-bp TaqI-PstI sequence and a 700-bp
PstI-StuI partial cDNA fragment into the
pBluescript-KS
vector and then cloned into the
EcoRI (filled in) site of the pSG5 mammalian expression
vector (31). The pSG-HOXB2 construct was obtained by an
EcoRI partial digestion of a 1700-bp genomic fragment cloned
into the EcoRI site of the pSG5 vector. We have already
described the pSG-HOXB3, pCT-HOXC6, and pSG-HOXD3 cDNA expression
vectors (27, 32).
Cell Culture and Transfection Assays--
NT2/D1 cells were
cultured in high glucose Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum (all from Life Technologies,
Inc.) in 5% CO2 in air-humidified atmosphere. NT2/C1 cells
were transfected by calcium phosphate co-precipitation as described
previously (32) with 5-8 µg of expression vector, 10-12 µg of
reporter plasmid, and 1-2 µg of either pRSV-
-gal or pPGK-
-gal
as internal control. Luciferase and
-galactosidase assays were
performed as described previously (32). All transfections were carried
out in duplicate batches, using at least two different DNA preparations
of each expression construct in at least three separate
experiments.
Preparation of Nuclear Extracts, DNase I Footprinting, and Gel
Retardation Assays--
Crude nuclear extracts from COS-7 transfected
cells were prepared 48 h after transfection as described by Dignam
et al. (33). Embryonic nuclear extracts were obtained from a
pool of 12.5 d.p.c. mouse embryos from which the posterior
(abdominal) part of the body had been surgically removed. Then the
remaining portion of the body was separated in two: the head (12.5 d.p.c. brain nuclear extracts) and the trunk (12.5 d.p.c. trunk nuclear
extracts). Crude nuclear extracts were then prepared from embryonic
tissues as described previously (32).
In order to perform DNase I footprinting experiments, the same
HinfI-HaeIII 534-bp fragment used for the
construction of pOTX2LucATTAbox-TK plasmid was cloned into a
HincII-SmaI-digested pGEM3 vector, and the
resulting construct was digested with SspI, end-labeled with a standard T4 polynucleotide kinase reaction, and cut at a
SacI site present in the polylinker. The resulting 455-bp
DNA fragment was purified by polyacrylamide gel electrophoresis and
then used for the DNase I footprinting assays as described previously
(34), in the presence of 2-8 µl of COS nuclear extracts or 0.5-2
µl of HOXB3 homeodomain (and immediately flanking residues, 4 amino acids at the N terminus and 10 amino acids at the 3' terminus) produced
in E. coli by a T7 promoter-based expression system, prepared as already described (32). 32P-Labeled 35-mer
double-stranded oligonucleotide HBE1
(5'-TATCCAGACTACTAATTAGGTGAAAATGATTACTG-3') was used as probe in
gel retardation assays as described previously (32) with 2-4 µl of
nuclear extracts. A 500-fold molar excess of the double-stranded HBE1
or HBE1m (5'-ATCCAGACTACTGCAGAGGTGAAAATGAT-3') oligonucleotides was
used for the competition assays. A polyclonal antiserum raised against
the bacterially produced HOXB3 homeodomain (32) was also used in gel
retardation assays.
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RESULTS |
HOXB1, HOXB2, and HOXB3 Transactivate a Mouse Otx2 5'-Flanking
Genomic Region in NT2/D1 Embryonal Carcinoma Cells--
Three
overlapping 1821-, 1310-, and 437-bp 5'-flanking DNA fragments
encompassing the major transcription start site of the mouse
Otx2 gene were cloned in front of the luciferase reporter gene in the pXP2 expression vector, and the resulting constructs were
named pOTX2Luc
1219, pOTX2Luc
710, and pOTX2Luc
+163,
respectively (Fig. 1A).

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Fig. 1.
Reporter and expression constructs used in
co-transfection assays. A, the reporter constructs
pOTX2Luc 1219, pOTX2Luc 710, and pOTX2Luc +163 were generated
by cloning three overlapping upstream genomic sequences of the mouse
Otx2 gene, extending from position 1219, 710, and +163,
respectively, to position +602 from the transcription start site,
upstream of the luciferase (LUC) coding region in the
pXP2Luc vector. The reporter construct pOTX2LucATTAbox-TK was
constructed by cloning in front of the minimal TK promoter (black
box) in the pT81Luc vector the Otx2 genomic sequence
from position 371 to +163. Panel B, full-length human or
mouse HOX cDNA sequences (HOX cDNAs) were cloned in
the mammalian expression vector pSG5, under the control of the SV40
early promoter (pSG-HOX). The arrows indicate
transcription start sites in all constructs.
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In order to assess if these 5'-flanking sequences are able to mediate a
regulation by HOX proteins, we co-transfected in NT2/D1 cells the
pOTX2Luc deletion constructs together with expression vectors encoding
HOXB1, HOXB2, and HOXB3. These Hox genes are known to be
already expressed at 7.5 d.p.c., when the concomitant transition
in Otx2 and Hox gene expression takes place (4). Full-length cDNAs coding for human HOXB1 and HOXB2 (35) were cloned
into the SV40 early promoter-based mammalian expression vector pSG5 to
generate the pSG-HOXB1 and pSG-HOXB2 constructs (Fig. 1B).
The pSG-HOXB3 plasmid has been previously described (32). In NT2/D1
cells, the reporter construct pOTX2Luc
1219 showed a 12-, 25-, and
9-fold increase in transcriptional activity when co-transfected with
pSG-HOXB1, pSG-HOXB2, and pSG-HOXB3, respectively. Likewise, the
construct pOTX2Luc
710 showed an 8-, 12-, and 6-fold increase
in transcriptional activity if co-transfected with pSG-HOXB1, -HOXB2,
and -HOXB3, respectively, while a higher level of basal transcriptional
activity was also noted (Fig.
2A). The pOTX2Luc
+163
construct, used as negative control, failed to show any increase in
transcriptional activity in co-transfection assays (Fig.
2A). The extent of the observed transactivation of the
reporter constructs was directly dependent on the concentration of the
HOX proteins used in the assay (data not shown).

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Fig. 2.
Transactivation of the mouse OTX2 Luc
constructs by the human HOXB1, HOXB2, and HOXB3 proteins. Panel
A, 10 µg of the pOTX2Luc 1219, pOTX2Luc 710, or
pOTX2Luc +163 reporter constructs were co-transfected in
NT2/D1 cells together with 5 µg of expression vectors encoding
HOXB1, HOXB2, or HOXB3. The open bars indicate the
basal activity of the three reporter constructs, while data from
co-transfection assays with HOX constructs are represented by
filled bars. Luciferase activity is expressed in -fold
induction, and the basal transcriptional activity is arbitrarily
considered equal to 1. pOTX2Luc 710 showed a 4-fold higher level of
basal transcriptional activity than pOTX2Luc 1219. Panel
B, the pOTX2LucATTAbox-TK reporter construct was co-transfected as
in panel A, but additional more posteriorly expressed
HOX-encoding constructs were used in the assay. Data are expressed as
in panel B.
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A close inspection of the DNA sequence surrounding the transcription
start site revealed the presence of several copies of the ATTA sequence
(Fig. 4), which is contained in most of the HOX/HOM-C target sites (8).
We then decided to clone the HinfI-HaeIII 534-bp
ATTA-rich sequence from position
371 to +163 in the pT81Luc expression vector (30) to generate the pOTX2LucATTAbox-TK construct, which contains also the thymidine kinase (TK) minimal promoter in front
of the luciferase reporter gene in order to increase its basal activity
(Fig. 1A). Co-transfection of the pOTX2LucATTAbox-TK with
the pSG-HOXB1, -HOXB2, and -HOXB3 showed a 15-, 17.5-, and 7-fold
increase in its basal transcriptional activity, respectively (Fig. 2B).
To assess the specificity of the transactivation on the Otx2
promoter of the anteriorly expressed Hox genes, expression
vectors for other Hox cDNAs (namely, HOXC6 and
Hoxd-8) were co-transfected in NT2/D1 cells together with
pOTX2LucATTAbox-TK, but they did not cause any increase in the
Otx2 promoter basal activity. We have already reported that
an efficient production of the proteins showing no effect on the
Otx2 promoter was achieved with the constructs used in the
present study (32). However, co-transfection with an expression vector
encoding for HOXD3, which belongs (together with HOXB3) to paralogy
group 3, caused an increase in the activity of the reporter construct
similar to that caused by HOXB3 (Fig. 2B). These results
indicate that the ability to transactivate the Otx2 promoter
is not a feature shared by all members of the Hox gene
family but is apparently common only to the anteriorly expressed
Hox genes, namely to those belonging to the paralogy groups
1, 2, and 3. Similar transfection experiments were also conducted in
HeLa and NIH3T3 cells, but no effect was observed on the
Otx2 promoter, suggesting that the cellular environment plays a crucial role in the transactivating activity of HOXB1, -B2, and
-B3 (data not shown).
HOXB1, HOXB2, and HOXB3 Bind to the Otx2 Promoter, but HOXB2
Differs in DNA Binding Specificity--
The binding of the HOXB1,
HOXB2, and HOXB3 proteins to the Otx2 promoter sequence was
tested by DNase I footprinting assay. Nuclear extracts were prepared
from COS-7 cells transfected with the pSG-HOXB1, pSG-HOXB2, and
pSG-HOXB3 constructs and tested on the Otx2 455-bp
SspI-HaeIII DNA fragment containing most of the
pOTX2ATTAbox-TK construct (Fig. 4). The only protection signal clearly
detected with HOXB1 and HOXB3 on these two probes was from position
177 and
186, respectively, to position
133 from the major
transcription start site (Fig.
3A). A weaker and shorter protection signal centered near position
180 was present with HOXB2-containing nuclear extracts. The protection was also confirmed on
the other DNA strand (data not shown). No other clear protections were
observed at the other ATTA sites present in the DNA fragment, indicating that (at least in the DNase I footprinting assay) a certain
degree of DNA binding specificity was achieved by HOXB1, HOXB2, and
HOXB3-containing nuclear extracts, although homeoproteins are known to
bind to virtually any DNA containing an ATTA core sequence in
vitro (8), suggesting that other determinants (i.e. PBX- or PBX-like factors) present in the COS nuclear extracts were able
to raise the in vitro DNA binding specificity of
HOX genes (41). Finally, an additional weaker protection
containing an ATTA core sequence and a hypersensitive site was found
near position
213 with HOXB1 only (Figs. 3A and
4). DNase I footprinting experiments
performed on the remaining 5' part of the pOTX2LucATTAbox-TK construct
did not reveal any other protection (data not shown).

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Fig. 3.
DNase I footprinting analysis of the binding
of HOXB1-, HOXB2-, and HOXB3-containing extracts to the ATTA-rich
Otx2 upstream region. Panel A, a 5'-end-labeled
455-bp SspI-HaeIII DNA fragment, corresponding
to positions 371 to +163 from the major transcription start site, was
incubated with 2, 4, and 8 µl of nuclear extracts from COS cells
transfected with HOXB1 (lanes 2-4) or HOXB2 (lanes
5-7), with 4 and 8 µl of HOXB3-containing (lanes
8-9) and mock-transfected (lanes 10-11) COS nuclear
extracts. Lane 1, no protein added to the reaction ( ).
Lanes 4 and 7, DNase I digestions were carried
out at a higher DNase I concentration. On the left,
bars represent the protected sequences; their position from
the major transcription start site and the proteins responsible for the
protection are also indicated. A hypersensitive site found only with
the HOXB1-containing extracts is also shown on the right.
Panel B, the same DNA fragment was incubated with 0.5, 1, and 2 µl of control (lanes 3-5) or HOXB3
homeodomain-containing (lanes 6-8) crude bacterial
extracts. Lane 1, G + A. Lanes 2 and
9, no protein added to the reaction ( ). A bar
on the left represents the protected sequence, and the
region not protected by the COS HOXB3-containing extracts is indicated
by a dashed line. Four DNase I-hypersensitive sites
(black arrows) are indicated on the right.
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Fig. 4.
Sequence of the upstream genomic region of
the mouse Otx2 gene from position 371 to +163 from the
major transcription start site. The entire Otx2 5'
genomic sequence cloned in the pOTX2LucATTAbox-TK construct is here
reported. The region bound by HOXB1, HOXB2, and HOXB3, as determined by
DNase I footprinting (Fig. 3), is boxed, and the HOXB2
binding site is stippled. The underlined sequence
is the HBE1 oligonucleotide used in gel retardation assays (see below).
The more upstream binding site found protected with HOXB1 only is also
boxed. The several unprotected ATTA consensus sites are
highlighted in boldface type. The SspI
restriction site used for the footprinting assay is indicated
above the sequence. An arrow indicates the major
transcription start site. Gray shading indicates the
sequence protected by the HOXB3 homeodomain.
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To confirm the presence of the protected DNA sequence on the
Otx2 genomic upstream sequence, we performed a DNase I
footprinting assay with a bacterially produced HOXB3 homeodomain
and immediately flanking residues (4 amino acids at the N terminus and
10 amino acids at the 3' terminus) on the 455-bp
SspI-HaeIII DNA fragment; a larger and strong
protection centered around the same site was detected from position
176 to position
92 (Figs. 3B and 4). Since it has been
reported that the protection signals found with homeodomains are
shorter than that obtained with entire proteins (32), we think that the
longer protection observed in our assay is due to the simultaneous
binding of several homeodomain molecules and that co-factors present in
COS nuclear extracts might inhibit HOX proteins from binding to
flanking DNA sequences. In fact, several DNA-hypersensitive sites were
also noticed, which presumably mark the borders of the DNA-bound
HOXB3-homeodomain (Fig. 3B). The HOXB3
homeodomain-containing bacterial extracts bind also to other sites
containing an ATTA core sequences, again showing that bacterial
extracts display a less stringent DNA binding specificity than COS
nuclear extracts (data not shown). The sequence of the entire AT-rich
region is reported in Fig. 4; the protected region is boxed
and the presence of unprotected ATTA sites is highlighted in
boldface type. The region protected by HOXB1 and HOXB3
shared a core sequence containing two palindromic TAATTA binding sites, whereas the site protected by HOXB2 contained a direct repeat of an
ACTT core sequence, quite divergent from the ATTA core consensus sequence (Fig. 4) but almost identical to the sequence (GCTTACTT versus ACTTACTT) found protected by HOXB2 on the
A
-globin enhancer (36).
On the basis of footprinting information, we designed a 35-mer
double-stranded oligonucleotide (HBE1, HOX-binding element 1;
underlined in Fig. 4) representing the sequence most
strongly protected by the nuclear extracts containing HOXB1 and HOXB3
but not HOXB2. As expected, nuclear extracts containing HOXB1 formed a
retarded DNA-protein complex with the HBE1 double-stranded
oligonucleotide (Fig. 5A, lane 4,
which was specifically competed by a
500-fold molar excess of unlabeled double-stranded oligonucleotide HBE1 but not by a comparable excess of unlabeled HBE1m double-stranded oligonucleotide, in which four bases inside the palindromic TAATTA site
have been mutagenized (Fig. 5A, lanes 5 and
6, and Fig. 6). Control
nuclear extracts from mock-transfected COS cells (Fig. 5A,
lanes 1-3) and HOXB2-containing nuclear extracts failed to show any specifically retarded complex (Fig. 5A, lanes
7-9). Similarly, HOXB3-containing COS nuclear extracts formed
multiple DNA-protein complexes, and at least three of them were
specifically competed by a 500-fold molar excess of HBE1 but not by
HBE1m (Fig. 5B, lanes 5-7), while the very faint
bands detected with the control extracts were not specific (Fig.
5B, lanes 1-3). In addition, an antiserum
against the HOXB3 homeodomain, which had already been shown to
interfere with the DNA binding capability of HOXB3 (32), was also able
to abolish the DNA binding activity of HOXB3-containing extracts on the
HBE1 site (Fig. 5B, lanes 5 and 8),
while the preimmune serum did not show any effect (data not shown). The addition to the binding reaction of the
-HOXB3 serum markedly improved the formation of unspecific DNA-protein complexes present in
the control extracts at very low amounts (Fig. 5B,
lanes 4 and 8).

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Fig. 5.
Gel retardation assays of the HBE1
double-stranded oligonucleotide by nuclear extracts from HOXB1-,
HOXB2-, and HOXB3-transfected COS cells. Panel A, nuclear
extracts from mock- (lanes 1-3), HOXB1- (lanes
4-6), and HOXB2-transfected COS cells (lanes 7-9)
were used in a gel retardation assay with the 32P-labeled
HBE1 double-stranded oligonucleotide. A 500-fold molar excess of
unlabeled HBE1 (lanes 2, 5, and 8) or
HBE1m (lanes 3, 6, and 9)
double-stranded oligonucleotides was also used. The specifically
retarded HBE1-protein complex is indicated on the left.
Lane 10, no proteins were added in the reaction. Panel
B, the same assay as in panel A was performed with
nuclear extracts from mock- (lanes 1-4) and
HOXB3-transfected COS cells (lanes 5-8). 1 µl of a
polyclonal anti-HOXB3 homeodomain antibody was added to the reaction
(lanes 4-8). The specifically retarded HBE1-protein
complexes (comp.) are indicated on the right.
C, no protein added to the reaction. F-, unbound
probe. A 3-day exposure and an overnight exposure were used for the
autoradiographs shown in panels A and B,
respectively.
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Fig. 6.
A mutation in the ATTA core sequence of the
HBE1 site abolishes the transactivation by HOXB1 and HOXB3 but not by
HOXB2. The reporter construct pOTX2LucATTAbox-TK was mutagenized
in the TAATTA core sequence (bottom part) of the HBE1 site
to generate the pOTX2LucATTAbox-TKm construct, and the extent of its
transactivation by the HOXB1, HOXB2, and HOXB3 proteins was compared
with the one of the wild type construct. Data are expressed in -fold
activation over the basal activity of the reporter construct
pOTX2LucATTAbox-TK (wt) or pOTX2LucATTAbox-TKm
(mut).
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Interestingly, sequence comparison of Otx2 5'-flanking
regions in mouse and Xenopus showed stretches of high
similarity in the 5' upstream region from position
371 to +163 and
nearby sequences,2 with 75%
identity in the footprinted sequence (Fig.
7). This level of identity is quite
remarkable for noncoding sequences from very divergent species,
suggesting that the function mediated by this region might be conserved
through evolution too and, thereby, be relevant for Otx2
gene regulation.

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Fig. 7.
DNA sequence comparison of the upstream
genomic region protected by HOX proteins in mouse and
Xenopus. Nucleotide residues identical in mouse
(M) and Xenopus (X) are indicated by a
bar. The footprinted sequence is boxed. An
overall 75% identity inside the footprinted sequence is found between
the two species. The dot indicates a nucleotide gap in the
murine sequence.
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HOXB1, HOXB2, HOXB3, and Nuclear Factors from Mouse Embryos Bind to
the Same Region in the Otx2 Upstream Regulatory Sequence--
To
assess whether HOXB1, HOXB2, and HOXB3 expressed in embryonic tissues
were able to recognize the same sequences bound by the nuclear extracts
prepared from COS HOXB1-, HOXB2-, and HOXB3-transfected cells, nuclear
extracts from 12.5 d.p.c. mouse embryos were prepared and used in
a footprinting assay. Both OTX2 and the three above mentioned HOX
proteins are expressed in the mouse at this developmental stage (11,
21, 37). The embryonic nuclear extracts from regions expressing
paralogy group 1-3 HOX proteins were prepared by surgical removal of
anterior limbs and all of the posterior (i.e. post-thoracic)
body. Subsequently, separation of the head from the rest of the trunk
was surgically achieved, and two separate preparations of nuclear
extracts were then performed. The resulting extracts were allowed to
react with the 455-bp Otx2 5' genomic fragment, where they
protected from DNase I digestion the same region recognized by HOXB1,
HOXB2, and HOXB3 (Fig. 8). No differences were observed between the DNA binding properties of the brain and trunk
extracts, suggesting that only anteriorly expressed Hox
genes were responsible for the binding activity found on this region
(Fig. 8). The 12.5 d.p.c. nuclear extracts were also able to bind
with a very strong affinity to the HBE1 site in a gel shift assay (data
not shown).

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Fig. 8.
DNase I footprinting analysis of the binding
of nuclear extracts from 12.5 d.p.c. mouse embryos to the
Otx2 upstream genomic region. The same 455-bp
SspI-HaeIIII probe used in Fig. 3 was tested in
a DNase I footprinting assay with 2 and 3 µl of nuclear extracts
prepared from 12.5 d.p.c. mouse embryonic trunks (lanes
2 and 3) and brains (lanes 4 and
5). Lanes 1 and 6, no protein ( ) was
added to the reaction. The arrows show two DNase
I-hypersensitive sites.
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HBE1 Mediates Transactivation by HOXB1 and HOXB3, but Not by HOXB2,
in Transfected Cells--
To test the role of the HBE1 site in the
transactivation of the Otx2 5' regulatory sequence by HOXB1,
HOXB2, and HOXB3, the ATTA core was mutagenized from TAATTA to
TGCAGA in the pOTX2LucATTA box-TK vector to generate the
pOTX2LucATTAbox-TKm construct (bearing the same mutation present in the
HBE1m oligonucleotide used for the competition experiments; see above),
which was then tested in a co-transfection assay together with
pSG-HOXB1, -HOXB2, and -HOXB3 in NT2/D1 cells. This mutation caused an
almost complete abrogation of the transactivation exerted by the HOXB1
and HOXB3 expression vectors. However, the extent of the
transactivation on the Otx2 promoter by HOXB2 was not
affected by the mutation at the HBE1 site (Fig. 6). These data show
that the HOXB1 and HOXB3 activity on the Otx2 promoter is
directly mediated by the HBE1 site. In contrast, the transactivation of
the Otx2 promoter by HOXB2 is not mediated by the HBE1 site,
in accordance with the lack of binding to the HBE1 element shown by
HOXB2 in vitro (Figs. 3A and 5A).
 |
DISCUSSION |
A huge amount of data during the last decade, including the
analysis of mouse knockout mutants, has demonstrated that positional information along the anteroposterior axis of the vertebrate trunk is
transduced by Hox genes, which encode a large family of
proteins evolutionary related to Drosophila HOM-C gene
products (1, 4). Recently, some of the molecules involved in the early
organization of the vertebrate rostral brain have also been identified,
and it has been suggested that they might play a role in the early patterning of the developing head in longitudinal and transverse domains (9). However, during development Hox genes are not expressed in the vertebrate rostral brain, whereas forebrain-specific genes are not expressed in the segmental portion of the trunk. Otx2 is present at 5.5-5.7 d.p.c. in the whole embryonic
portion of the epiblast, while at later stages of development (7.0-7.5 d.p.c.) it becomes progressively restricted to the anterior portion of
the embryo, mainly the neuroectoderm of the headfold. In contrast, the
expression of Hox genes begins to appear in the posterior part of the epiblast and progressively moves to more anterior regions.
Thus, Otx2 and Hox genes show a complementary
spatial regulation during embryogenesis. Moreover, Otx2 and
Hox genes have been shown to be down- and up-regulated,
respectively, by retinoic acid both in vivo and in
vitro (11, 21, 23). How Hox and Otx2
patterning information can complement each other during embryogenesis
and how their mutually exclusive expression patterns are established
still remain unsolved questions. In principle, it was possible that
HOX genes mediated the Otx2 restriction to the
anterior third of the embryo through down-regulation of Otx2 expression in the posterior two-thirds. If this was the case, it was
also possible that in vertebrates RA acts by activating Hox
gene expression, which in turn would repress Otx2 expression by a direct interaction with its cis-regulatory sequences.
On the contrary, we provide here evidence that Otx2 is,
indeed, a candidate target for regulation by HOX proteins, but anterior HOX proteins activate, rather than repress, Otx2
transcription.
In fact, a 1821-bp 5' genomic fragment of the Otx2 promoter
is positively regulated by co-transfection with the expression vectors
for HOXB1, HOXB2, and HOXB3 in an embryonal carcinoma cell line
(NT2/D1), and a shorter fragment of only 534 bp is able to drive this
regulation. HOXB1 and HOXB3 bind to a sequence containing two
palindromic TAATTA sites, which bear four copies of an ATTA core
sequence, a common feature of most HOM-C/HOX binding sites (8), while
HOXB2 binds to an adjacent direct ACTT repeat. Mutagenesis of the
TAATTA motif abolished HOXB1- and HOXB3-mediated, but not HOXB2-mediated, transactivation. These findings indicate that Hox genes in NT2/D1 cells up-regulate the expression of the
Otx2 gene and that a direct role for Hox genes in
the down-regulation of the early Otx2 expression in the
posterior mouse embryo is unlikely. Moreover, a repressive role for
HOXB1, HOXB2, and HOXB3 proteins is unlikely, because these
homeoproteins have already been shown to act as transcriptional
activators on other cis-regulatory sequences (32, 36, 38,
39). However, we cannot rule out the possibility that other
determinants or co-factors necessary for the repression of the
Otx2 promoter might be absent (or present in a limiting
amount) in the NT2/D1 cells used in the transfection assay.
The requirement for an inductive signal from anterior mesendoderm to
stabilize Otx2 expression in the ectodermal layer has already been reported (14, 21). In particular, it has been shown that
isolated explants from mouse ectoderm become committed to express
Otx2 in a cell-autonomous fashion only by the midstreak stage of development, corresponding to the time of onset of
Hox gene expression (21). These findings have been
implemented by data from homozygous mutant Otx2
/
mice showing that the inductive signal from the
mesendoderm is dependent upon a functional Otx2 allele. In
fact, lacZ expression in Otx2
/
mouse embryos, sustained by the vector used in the disruption experiment, was strongly maintained in the external layer, the endodermal component of the mesendoderm, but was almost undetectable in
the embryonal ectoderm (17). It is conceivable that Hox
genes might increase the Otx2 expression in the mesendoderm
up to a threshold value necessary to achieve a proper inductive signal on the overlying ectoderm. This hypothesis is further supported by the
observation that the Otx2 expression becomes progressively stronger as its restriction to the anterior neuroectoderm takes place.
Although in the NT2/D1 embryonal carcinoma cell line it is possible to
reproduce the complementary transcriptional regulation of
Otx2 and Hox gene expression by retinoids, we
still do not know if these cells indeed provide an environment truly
equivalent to the one found in the developing embryo. However, very
recently Kimura et al. (40) identified a 49-bp Otx2
cis-regulatory element that is necessary and sufficient to drive a
transgene expression in the cephalic mesenchyme of the developing
mouse. In addition, they identified in pufferfish Fugu
rubripes an Otx2 cis-regulatory sequence functionally
equivalent to the murine 49-bp element and performed a mutational
analysis of the murine genomic sequences that were most conserved
between mouse and pufferfish. In this manner, the authors found that
only mutagenesis of the same TAATTA core sequences identified in
this report (Fig. 9) leads to a complete loss of transgene expression in the developing mouse embryo; these data
demonstrate that the TAATTA motifs identified in our DNA binding and
mutational analysis play a relevant functional role not only in
vitro, but also in vivo.

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Fig. 9.
Comparison of mutagenized sequences in Ref.
40 and our work. Mutation of the TAATTA motif in the murine
Otx2 5' cis-regulatory sequence (wt)
abolishes the transgene expression in the cephalic mesenchyme of the
mouse embryo (Ref. 40; top row), and the
transactivating activity of HOXB1 and HOXB3 in NT2/D1 cells (this
work; bottom row).
|
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The observation that Hox genes (namely, HOXC6 and
Hoxd-8) that, at later stages of development, will be
expressed in more posterior domains are not able to transactivate the
Otx2 promoter shows that such interaction is not a common
feature of all homeodomain-containing genes but rather is restricted
only to the most anteriorly expressed.
HOXB1, HOXB2, and HOXB3 are all able to bind to the same region in the
Otx2 promoter but significantly differ in DNA binding specificity. HOXB1 and HOXB3 bind to the same site, with minor differences in the length of the protected sequence, whereas the adjacent site protected by HOXB2 contained a direct repeat of an ACTT
core repeat, quite divergent from the ATTA consensus sequence. An
almost identical sequence (GCTTACTT versus ACTTACTT) was
found protected in the A
-globin enhancer by an activity
specifically found in the K562 cell line, afterward identified as HOXB2
(36).
Finally, the data here presented indicate that, despite a well
documented lack in DNA binding specificity shown by HOM/Hox genes in vitro (4), the HOXB1, HOXB2, and HOXB3-containing COS nuclear extracts (but not the HOXB3-containing bacterial ones) show
some in vitro specificity, because in the genomic fragment used in the footprinting assay many other ATTA sites are present that
are not recognized by any of the three homeoproteins. The DNA binding
specificity shown by COS nuclear extracts might be due to determinants
present in the unpurified nuclear extracts such as, for example, PBX or
PBX-like proteins (41). The same DNA binding specificity is shared by
nuclear extracts prepared from 12.5 d.p.c. mouse embryos, showing
that the COS nuclear extracts can be considered quite representative of
the in vivo situation. Moreover, the relevance of the
protected sequence within the Otx2
371 to +163 region is
also supported by the striking homology (75% identity) that we found
between species as divergent as mouse and Xenopus, even
higher than the reported homology between the pufferfish and mouse
genomic sequences (40). It is here worth noting that we cannot
establish if quantitative differences in DNA binding shown by the three
homeoproteins used in this study are due to minor differences in the
rate of production in COS cells or in their DNA binding affinity.
In conclusion, we have shown that HOXB1, HOXB2, and HOXB3 are able to
positively interact with the Otx2 upstream regulatory sequence in an embryonal carcinoma cell line, and we have characterized their DNA-binding sites on this promoter; HOXB1 and HOXB3 bind to
similar sequences, while HOXB2 displays a completely different DNA
binding specificity. The region bound by the three homeoproteins is
strikingly conserved through evolution and necessary (at least for
HOXB1 and HOXB3) to mediate the regulation of the Otx2
promoter. Taken together, our data support the hypothesis that
Hox genes might play a role in the refinement of the
Otx2 early expression boundaries in vivo.
We are deeply indebted to Dr. M. E. Bianchi for helpful discussions, critical reading of the manuscript and
continuous support. We also thank Dr. A. Mallamaci and Dr. M. G. Giribaldi for sharing unpublished sequences, Dr. F. Mavilio for
critical reading of the manuscript, and M. Sottocorno for secretarial
work.