From the DIBIT-Istituto Scientifico H. San Raffaele,
Via Olgettina 58, 20132 Milano, the
§§ Department of Biomedical Sciences, University
of Modena School of Medicine, Via Campi 287, 41100 Modena, Italy,
the ¶ Division of Developmental Neurobiology, National Institutes
for Medical Research, The Ridgeway, Mill Hill,
London NW7 1AA, United Kingdom, and the
Stowers Institute for Medical Research,
Kansas City, Missouri 64110
Received for publication, December 12, 2000, and in revised form, February 23, 2001
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ABSTRACT |
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Regionally restricted
expression patterns of Hox genes in developing embryos rely
on auto-, cross-, and para-regulatory transcriptional elements. One
example is the Hoxb1 auto-regulatory element (b1-ARE), which drives expression of Hoxb1 in the fourth rhombomere
of the hindbrain. We previously showed that HOXB1 and PBX1 activate
transcription from the b1-ARE by binding to sequences required for the
expression of a reporter gene in rhombomere 4 in vivo. We
now report that in embryonal carcinoma cells, which retain
characteristics of primitive neuroectodermal cells, the b1-ARE displays
higher basal and HOX/PBX-induced activities than in other cell
backgrounds. We have identified a bipartite-binding site for SOX/OCT
heterodimers within the b1-ARE that accounts for its cell
context-specific activity and is required for maximal transcriptional
activity of HOX/PBX complexes in embryonal carcinoma cells.
Furthermore, we found that in an embryonal carcinoma cell background,
HOXB1 has a significantly higher transcriptional activity than its
paralog HOXA1. We map the determinants for this differential activity within the HOXB1 N-terminal transcriptional activation domain. By using
analysis in transgenic and HOXA1 mutant mice, we extended these
findings on the differential activities of HOXA1 and HOXB1 in
vivo, and we demonstrated that they are important for regulating aspects of HOXB1 expression in the hindbrain. We found that mutation of
the SOX/OCT site and targeted inactivation of Hoxa1 both
impair the response of the b1-ARE to retinoic acid in transgenic
mice. Our results show that Hoxa1 is the primary mediator
of the response of b1-ARE to retinoic acid in vivo
and that this function is dependent on the binding of SOX/OCT
heterodimers to the b1-ARE. These results uncover novel functional
differences between Hox paralogs and their modulators.
The HOX homeodomain-containing transcription factors control cell
fate and developmental patterns in all metazoans, leading to the
generation of morphological differences along body axes (reviewed in
Ref. 1). In most vertebrates the four Hox clusters encode 39 distinct proteins in which the homeodomain
(HD)1 and flanking amino
acids dictate the DNA binding specificity by recognizing a restricted
set of sites containing the core consensus sequence TNAT(G/T)(G/A)
(2-4). Despite the apparent similarity in consensus DNA recognition
sites, HOX proteins can modulate their binding properties and
specificity through the concomitant activity of an emerging array of
cofactors. Interactions with cofactors such as the HD-containing
proteins of the EXD/PBX (PBC) (3-6) and MEIS/PREP (MEINOX) (7-11)
families can modulate the affinity and stability of DNA binding and
regulate transcriptional activity of HOX proteins.
Transgenic analysis in mice has led to the identification
of sequences that function as HOX target sites in vivo for
the auto-, cross-, and para-regulatory interactions among
Hox genes (6, 12-15). Several of these in vivo
target sites are composed of bipartite and overlapping HOX- and
PBX-binding motifs (HOX/PBC sites) and represent useful models for
analyzing the function of HOX-containing transcriptional complexes.
Protein-protein contacts involved in functional interactions between
the HOX and the EXD/PBX proteins were found to be mediated by both the
N-terminal region of the HD (16) and the short conserved hexapeptide or
YPWM motif (17-19), located upstream of the HD in a subset of HOX
proteins (paralogy groups 1-8). Recent crystal structure analysis of
HOX/PBX homeodomain complexes bound to DNA target sites with relevance
in vivo have provided a useful model for understanding how
these interactions can influence specificity of binding (20, 21).
The identification of bona fide Hox-responsive
enhancers and promoters allowed analysis of the transcriptional
properties of these proteins. These studies have shown that HOX
proteins share the modular type structure of most eukaryotic
transcription factors, featuring separate DNA binding and activator or
repressor domains (5, 22-26). One of the HOX-binding elements examined in most detail in vivo is contained in a highly conserved
auto-regulatory enhancer of the Hoxb1 gene (b1-ARE). This is
a key cis-regulatory element for the normal rhombomere 4 (r4)-restricted expression of Hoxb1 in the developing
hindbrain. Genetic analysis has revealed that in an early phase
Hoxa1 and Hoxb1 function synergistically to
establish initial r4 identity by triggering the auto-regulatory loop
(6, 27, 28). As a part of this process, the early activation of
Hoxb1 and Hoxa1 themselves is directly mediated by RA signaling through the presence of RA response elements (RAREs) located at the 3' ends of both genes (28-31). In later stages
Hoxb1 is required to maintain r4 identity (27, 28). Even
though the b1-ARE does not contain a canonical RARE, it indirectly
participates in mediating the RA-dependent ectopic
activation of Hoxb1 in anterior regions (r2), through its
auto-regulatory ability (6).
Auto- and para-regulation of Hoxb1 are dependent on the
cooperative binding of HOXB1 or HOXA1, and a member of the PBX family on three conserved sequence motifs (R1, R2, and R3) in the b1-ARE related to a consensus bipartite HOX/PBC-binding site (5, 6). Each of
these HOX/PBX repeats contributes to the r4-restricted expression of a
reporter gene in transgenic mice and to the indirect RA response. The
R3 motif, however, makes the largest contribution to the b1-ARE
regulatory activity (6). Interestingly, the b1-ARE is not active in
other regions of the embryo where Hoxb1 and Pbx1 are coexpressed (spinal cord, paraxial mesoderm, endoderm, and limb
buds), suggesting that region-specific expression in the hindbrain
might be determined by differential activity of additional factors.
We reported previously (5) that the human HOXB1 and PBX1 proteins
cooperatively activate transcription from a basal promoter under the
control of the b1-ARE in transfected mammalian cells. Comparing the
functional selectivity of proteins from a range of Hox
paralogy groups, we showed (5) that only a restricted subset of HOX
proteins (HOXA1, HOXB1, and HOXB2) are able to activate transcription
from the b1-ARE in cooperation with PBX1. Selective recognition of the
R3 motif by the HOXB1/PBX1 complex is mediated by the N
terminus of the HOX homeodomain, whereas the major transcriptional activator domain is provided by the HOXB1 N-terminal region (5, 26).
In this study we show that the basal and HOX/PBX-induced
transcriptional activities of b1-ARE, in comparison to other cell lines, are significantly higher in murine or human embryonal carcinoma (EC) cells, which retain phenotypic and molecular characteristics of
primitive neuroectodermal cells (32, 33). In this context, HOXB1
displays a stronger transcriptional activation than its paralog HOXA1,
in cooperation with PBX1. The differential activity between HOXB1 and
HOXA1 is not based on the DNA-binding properties of the HOXB1/PBX1
complex but rather on specific determinants within the HOXB1
transcriptional activation domain that selectively interact with the
transcriptional machinery. Furthermore, we show that the full
transcriptional activity of HOX/PBX heterodimers on the b1-ARE is
dependent on the binding of a SOX/OCT heterodimeric complex to a
bipartite site located immediately upstream of the R3 sequence.
Mutation of this site, as well as the targeted inactivation of the
Hoxa1 gene, impairs the response of the b1-ARE to RA
treatment in transgenic mice and the establishment of a full ectopic
auto-regulatory circuit of Hoxb1 in vivo. Our
results show that the product of the Hoxa1 gene is required
for the response of Hoxb1 to RA, and this activity requires
a functional SOX/OCT-binding site within the b1-ARE. Together, these
results have uncovered novel differences among HOX paralogs and their modifiers.
Expression Vectors and Reporter Plasmids--
All expression
constructs are derivatives of the SV40 promoter-based expression vector
pSG5 (34). HOXB1, HOXA1, HOXB2, and PBX1 expression vectors were
described previously (5). pSGB1/A1HD was generated by swapping the
region spanning the FDWM motif and the homeodomain of HOXB1 (aa
175-257) with the corresponding region of HOXA1 (aa 206-289), whereas
pSGA1/B1CT was generated by substituting the C-terminal region of HOXA1
(aa 290-336) with that of HOXB1 (aa 258-296). The mutated cDNAs
were cloned into the BamHI site of pSG5. pGAL1-147 (35)
contains the DNA binding domain of yeast GAL4 (amino acids 1-147).
pGALVP16 was generated by cloning an PCR-amplified region encompassing
the C terminus (last 80 aa) of the VP16 protein in frame with the
GAL1-147 protein at the BamHI site of the pGAL1-147
vector. PCR was carried out with Pfu polymerase
(Stratagene). All PCR-generated fragments were sequenced on both
strands, and expression of all proteins was preliminarily tested using
a T7 polymerase-based transcription and reticulocyte lysate-based
translation system (Promega).
The luciferase reporter construct pMLluc is a pXP2-based vector (36)
containing the adenovirus major late basal promoter (from
For transgenic mouse analysis, the wild type Hoxb1
5'-flanking StuI-HindIII fragment containing the
b1-ARE was cloned into the lacZ basal reporter vector BGZ40
(38) as described previously (6). Point mutations in the
SOX/OCT-binding sequence (TAAT to CCGG and TTTGTC to CGCTGT) were
generated by site-directed mutagenesis in M13 or inverse PCR, followed
by double-strand sequencing.
Cell Culture and Transfection--
COS-7 cells were maintained
in Dulbecco's modified Eagle's medium, supplemented with 10% fetal
calf serum (Life Technologies, Inc.), 100 IU/ml penicillin, and 100 µg/ml streptomycin. P19 and NT2/D1 cells were maintained in
Electrophoretic Mobility Shift Assay (EMSA)--
To obtain total
cell protein extracts, cells were collected from confluent plates,
washed with phosphate-buffered saline, pelleted, frozen with liquid
nitrogen, and lysed by resuspension in 5 volumes of Extraction Buffer
(10 mM Hepes, pH 7.9, 0.4 M NaCl, 0.1 mM EDTA, 0.5 mM DTT, 5% glycerol, 0.5 mM phenylmethylsulfonyl fluoride, 1% Trasylol). The lysate
was then centrifuged for 30 min at 34,000 rpm in a Beckman Ti-50 rotor,
and the supernatant was stored in aliquots at Generation and Analysis of Transgenic Mice--
Transgenic mice
were produced by microinjection of DNA into fertilized eggs from
crosses between F1 (CBA × C57) females and males. Whole mount
The Transcriptional Activity of the HOXB1/PBX1 Complex Is Maximal
in a Neuroectodermal Cell Background--
We have shown previously
that the HOXB1/PBX1 heterodimer is able to activate transcription of a
reporter gene under the control of the b1-ARE in mammalian cell lines
(5, 26). To investigate the influence of the cell context on this
system, we compared the transcriptional activity of the HOXB1/PBX1
complex in different cell lines, including COS-7, HeLa, NIH3T3, P19,
and NT2/D1 cells. The murine P19 and the human NT2/D1 EC cells are
originally derived from germ cell tumors and retain many
characteristics of primitive neuroectodermal cells, including the
ability to differentiate into neurons (32, 33) and activate the four
Hox gene clusters upon induction with retinoic acid
(43-45). To measure transcriptional activity, reporter constructs were
made by placing a luciferase gene under the control of the adenovirus
major late basal promoter either alone (pAdMLluc) or in combination
with the 150-bp b1-ARE Hox-responsive region (pAdMLARE). These were
cotransfected with expression constructs for HOXB1 and PBX1 into the
various cell lines and assayed for reporter activity (Fig.
1). In COS-7, HeLa, or NIH3T3 cells the
basal activity of the reporters with and without the b1-ARE enhancer
were identical (Fig. 1 and data not shown). In contrast, in both P19
and NT2/D1 EC cells the construct containing the b1-ARE enhancer
(pAdMLARE) displayed a 7-fold higher basal level, compared with the
control (pAdMLluc). In non-EC cells (e.g. COS-7 in Fig. 1),
coexpression of HOXB1 and PBX1 led to a 8-fold transactivation of the
pAdMLARE reporter. In P19 and NT2 cells, however, coexpression of HOXB1
and PBX1 led to significantly higher levels of transactivation, ~20-
and ~60-fold, respectively, over the pAdMLARE basal activity of the
reporter, corresponding to a ~140- and ~360-fold increase in
activity, respectively, of the enhancer-less pAdMLluc reporter (Fig.
1).
Transfection of the PBX1 expression vector alone had no effect on
reporter activity in any cell context, whereas transfection of HOXB1
alone led to a 2-3-fold activation only in EC cells presumably through
interaction with the endogenous levels of PBX1 expressed in these
cells. The higher activity of the HOXB1/PBX1 complex on the b1-ARE
target was found not to be due to a higher transfection efficiency in
EC cells, as the percentage of transfected cells was found to be
comparable to that of the other cell lines (data not shown).
Furthermore, we observed no broad differences in the capability of EC
versus COS-7 cells to sustain transcriptional activation
(see "Experimental Procedures"). Our findings that the basal
activity and the ability of the HOXB1/PBX1 complex to act on the b1-ARE
are much higher in EC cells, compared with other cell lines, suggest
either the presence of EC-specific enhancing factors or COS-7
inhibiting components that modify the b1-ARE regulatory potential.
HOXB1 and HOXA1 Differentially Activate Transcription through the
b1-ARE in Neuroectodermal Cells--
We previously reported that only
HOX proteins belonging to paralogous groups 1 and 2 can bind and
activate the full b1-ARE or multimerized versions of R3 in cultured
cells in a PBX-dependent manner (5). To compare directly
the activity of different HOX/PBX complexes in an EC cell background,
expression constructs for HOXA1, HOXB1, and HOXB2 were cotransfected in
P19 cells, together with the reporters containing the entire b1-ARE
(pAdMLARE) or a trimer of the R3 motif (5). The basal activity of
pAdMLARE was higher than that of pAdMLR3 and was only weakly induced by cotransfection with HOX proteins alone (Fig.
2). On the pAdMLR3 reporter, consistent
with our previous results using non-EC cells, the activities of the
HOXB1/PBX1 and the HOXA1/PBX1 complexes were comparable, resulting in
an 80-100-fold transactivation. The HOXB2/PBX1 complex was also active
although it remained lower (15-fold transactivation) (Fig. 2). In
contrast, on the full b1-ARE reporter (pAdMLARE) while HOXB1 and PBX1
induced a 20-fold transactivation (see also Fig. 1), surprisingly HOXA1
only weakly activated (6-fold) the reporter expression in combination
with PBX1 (Fig. 2). HOXB2 displayed an even lower (4-fold) activity
with PBX1 (Fig. 2). These results indicate that in the EC cell
background HOXB1 activates transcription more efficiently than its
paralog HOXA1 in cooperation with PBX1 through the entire b1-ARE
element. This differential activity was never observed in
non-neuroectodermal cell lines.
As the use of a multimerized version of the HOX/PBC repeat 3 target
sequence apparently relaxes the paralog selectivity, allowing HOXA1 and
HOXB2 to function as effectively as HOXB1, we considered the
possibility that other enhancer sequences within the b1-ARE might
influence the activity of the different HOX/PBX heterodimers. In order
to analyze the contribution of different regions of the enhancer in
restricting transcriptional activation, we cotransfected reporter
constructs containing sequential 5' deletions of the b1-ARE (Fig.
3B) with HOXB1, HOXA1, and
PBX1 in P19 cells. In deletions with increasing size there was a
progressive reduction in the overall level of transactivation that
progressively reduced expression to 70, 50, and 30% of the entire
b1-ARE (Fig. 3A). However, the differential ability of
HOXB1/PBX1 versus HOXA1/PBX1 to activate these sites was
maintained even on a reporter containing only repeat 3 and its
surrounding sequences (ARE A SOX/POU-OCT-binding Site Is Necessary for Full Transcriptional
Activity of the b1-ARE in EC Cells--
In the b-1ARE, located between
the R2 and R3 HOX/PBC sites, there is a bipartite sequence motif that
is highly conserved in the human, mouse, chicken and pufferfish
Hoxb1 locus (6). In this motif (designated SOct
in Fig. 4), the 5'-most part (TCTTTGTC) closely resembles the target sequence for the HMG box protein SOX-2
(46-48), whereas the 3' part (ATGCTAAT) shows a high degree of
similarity with the consensus recognition sequence for
POU/Octamer-binding proteins (49). To test the role of this putative
SOX/OCT heterodimer-binding site in the function of the b1-ARE in EC
cells, we generated a 11-bp mutation (ARESOctm) encompassing both the
SOX and the POU/OCT sites in the context of the whole b1-ARE sequence
(Fig. 4B). In cotransfection experiments this SOX/OCT site
mutation reduced transactivation of the HOXB1/PBX1 complex by 50% and
that of HOXA1-PBX1 by 77% (Fig. 4A). Interestingly, this
SOX/OCT mutation completely abolished the EC cell-specific basal
activity of the b1-ARE, as well as the weak but detectable
transactivation by HOXB1 in the absence of PBX1 (Fig. 4A).
To help distinguish between the contribution of the SOX-specific
versus the POU/OCT-specific sequence on the b-1ARE activity,
an additional mutation (ARE
These results indicate that the increased basal activity of the b1-ARE
in EC cells compared with COS cells is due to EC-specific or
EC-enriched factors interacting with the SOX/OCT site and that the
SOX-specific hemisite is necessary for this activity. The same factors
appear to affect the overall transcriptional activity of HOX/PBX
heterodimers on the b1-ARE through this site. Furthermore, our data
show that transactivation by HOXA1/PBX1 is more dependent upon the SOct
motif than that by HOXB1/PBX1.
The SOX/OCT Site Is Cooperatively Bound by SOX-2/OCT1 and
SOX-2/OCT3/4 Heterodimers--
Since the conserved SOX/OCT site has a
functional role in the b1-ARE enhancer activity in EC cells, we looked
for EC cell-specific binding activities on this site by EMSA. The
octamer-binding proteins OCT1 and OCT3/4 and the HMG box protein SOX-2
are expressed at high levels in EC cells and were previously shown to
bind and regulate cooperatively the activity of developmentally
regulated enhancers such as that of FGF-4 (50, 51). Hence, we used a double-stranded oligonucleotide spanning the SOX/OCT site of the FGF-4
enhancer as a control. The b-1ARE EMSAs were carried out using as probe
a double-stranded oligonucleotide containing the SOX/OCT site (SO-ARE)
in combination with total cell extracts obtained from either P19 or NT2
EC cells (Fig. 5). Except where indicated, poly(dG-dC) was used instead of poly(dI-dC) as nonspecific DNA competitor to allow binding by HMG box proteins (50). As shown in
Fig. 5 (lanes 2 and 6), in both P19 and NT2/D1
cell extracts a major SOX-2/OCT3/4 complex and a minor SOX-2/OCT1
complex bind the b1-ARE probe. These were specifically competed and/or
supershifted by the addition of ant-OCT1 and anti-OCT3/4 antibodies to
the binding reaction (Fig. 5, lanes 3 and 4 and
7 and 8). Addition of poly(dI-dC) competed out
the binding of the heterodimeric complexes and allowed the formation of
monomeric OCT1 and OCT3/4 complexes (Fig. 5, lanes 5 and
9).
In comparison, at least five complexes with similar intensity were
detected on the control FGF-4 probe, and they correspond to the
individual monomers and the SOX-2/OCT1 and SOX-2/OCT3/4 heterodimers
(Fig. 5, lanes 11 and 15). The identity of all
complexes was confirmed by anti-OCT1 and anti-OCT3/4 antibodies (Fig.
5, lanes 12 and 13 and 16 and
17), and again the addition of poly(dI-dC) allowed only
binding of the monomeric OCT complexes (Fig. 5, lanes 14 and
18). These results indicate that OCT1 and OCT3/4 bind
exclusively as heterodimers with SOX-2 to the SOX/OCT sequence in the
b1-ARE.
To check whether binding of the SOX-2/OCT complexes on the b1-ARE
SOX/OCT site also occurs in embryonic tissues, we used total cell
extracts obtained from the central nervous system of 9.5-dpc mouse
embryos. As shown in Fig. 6 (left
panel), strong binding of both SOX-2/OCT3/4 and SOX-2/OCT1
complexes to the b1-ARE sequence was observed in embryo extracts, with
a pattern similar to that observed with EC cell extracts. The identity
of the complexes was also confirmed with anti-OCT and anti-SOX-2
antibodies and by the differential use of poly(dG-dC) or poly(dI-dC) as
nonspecific competitors (Fig. 6). A much weaker binding was observed on
the FGF-4 probe, particularly for the SOX-2/OCT3/4 complex (Fig. 6, right panel). These results show that the b1-ARE sequence
binds protein complexes containing SOX and OCT proteins present in
embryonic tissues at a stage corresponding to the maximal activity of
the b1-ARE in the developing hindbrain (6).
SOX-2 and OCT1 Cooperatively Enhance the Activity of the b1-ARE in
COS Cells--
Taken together, the experiments above suggest that the
different activity of the b1-ARE in EC cells compared with COS cells could be mediated by differences in the availability of SOX and OCT
proteins. To assess this issue directly, we cotransfected expression
plasmids for the SOX-2, OCT1, and OCT3/4 into COS-7 cells together with
the b1-ARE reporter in the presence or absence of HOXB1 and PBX1 (Fig.
7). No activation of reporter expression was observed by transfection of any of the individual plasmids in the
absence of HOXB1 and PBX. Cotransfection of SOX-2 and OCT1 caused a
4-fold induction of the reporter basal activity bringing it closer to
that observed in EC cells. More important, the activity of SOX-2/OCT1
had an additive effect on that of the HOXB1/PBX1 complex on the b1-ARE
and caused a further 2-3-fold increase in its transcriptional activity
(Fig. 7). OCT1 alone, but not SOX-2, had some effect on the activity on
the HOXB1/PBX1 complex, whereas OCT3/4 had no effect, neither alone nor
in combination with SOX-2. These results confirm that the SOX/OCT site
within the b1-ARE significantly contributes to its enhancer activity by
recruiting SOX/OCT heterodimers in the appropriate cell context that
are able to increase the transcriptional activity of HOX/PBX
complexes.
The SOX/OCT-binding Site Is Necessary for Ectopic Activation of
b1-ARE by Retinoic Acid in Vivo--
In view of our data indicating
that the SOX/OCT-binding sequence flanking R3 is important both for the
basal activity of the b1-ARE and for the activation by HOX proteins in
cell culture, we investigated the role of these sequence using
transgenic analysis. Deletions 3' of R3 had no effect on the in
vivo regulatory activity of the enhancer (data not shown). Two
additional mutants were generated in the SOct sites in the context of a
highly conserved 331-bp StuI/HindIII fragment
containing the b1-ARE (Fig. 8). In the
first case, where we introduced a mutation (TAAT to CCGG) into the more
3' OCT-specific hemisite, lacZ reporter expression appeared
normal (Fig. 8). At 9.5-10.0 dpc reporter staining was correctly
restricted to r4 and second arch neural crest in embryos not exposed to
RA and ectopically induced in r2 in all embryos (n = 10) that had been exposed to RA at an earlier time (Fig. 8,
A-D). This is identical to the properties of the wild type b1-ARE and shows that mutation of the POU homeodomain-binding sequence
is still compatible with full enhancer activity. In contrast, a second
variant that alters both the OCT (TAAT to CCGG) and the SOX (TTTGTC to
CGCTGT)-binding hemisites completely abolished (4/5 embryos), or
dramatically reduced (1/5 embryo), the RA-induced ectopic stripe of
reporter expression in r2 of embryos exposed to RA (Fig. 8,
E-G). The same mutation had no obvious effect (7/7 embryos)
on normal r4 expression (Fig. 8, E Hoxa1 Is Required for RA-induced Ectopic Activation of b1-ARE in
Vivo--
Our results obtained by EC cell transfection indicate that
the transactivation of the b1-ARE by a HOXA1/PBX1 complex is weaker and
more dependent on the presence of a SOct site as that of the HOXB1/PBX1
complex. This differential activity, given that the response of the b1
ARE to retinoids in embryos requires the SOct site, might reveal HOXA1
as being the primary component involved in mediating the response of
Hoxb1 to RA in vivo. To test this idea, we crossed a
transgenic reporter line including the b1-ARE (31) into a
Hoxa1 mutant genetic background (52) and assayed its
response to RA in mutant and wild type embryos. This
Hoxb1/lacZ transgene does not contain the 3'-RAREs
responsible for Hoxb1 activation in the ectoderm (28, 31)
and in the endoderm (53), and therefore any response to RA would be
indirect. Untreated wild type and heterozygous
Hoxa1+/
Our results in vivo indicate that the ectopic activation of
this element by RA requires both the presence of a functional SOX/OCT
site and the presence of a functional Hoxa1 gene. This implies that for HOXA1 to function efficiently on the b1-ARE, it is
important that SOX and OCT proteins are recruited to the SOct site. It
is interesting that even though HOXA1 and HOXB1 both participate in
regulating normal r4 expression (27, 28), we find that, unlike the RA
response, transgene expression is maintained in r4 despite mutation of
the SOct site. This suggests that in vivo as in the EC
cells, HOXB1 is not as dependent as HOXA1 upon SOX and OCT proteins and
is able to activate the b1-ARE in r4.
Determinants for the Differential Activity of HOXB1 and HOXA1
Reside within the HOXB1 N-terminal Domain--
To examine whether the
differential activation of HOXA1 and HOXB1 on the b1-ARE is related to
intrinsic structural differences between these proteins, we used a
domain swap approach. The HOXA1 and HOXB1 proteins have almost
identical homeodomains (54), and they share a conserved FDWM motif just
N-terminal to the homeodomain that serves as the HOX/PBX interaction
surface, necessary for cooperative DNA binding and transcriptional
activation (reviewed in Ref. 3). HOXA1 and HOXB1 bind the b1-ARE R3
site with the same affinity in vitro and have similar
transcriptional activity on the full b1-ARE element in non-EC cells and
in transgenic mice (5, 6, 55). However, the two proteins differ
completely in their C-terminal and N-terminal regions (56), and for
HOXB1 the N-terminal region contains the major transcriptional
activation domain used in the context of heterodimers with PBX1 (5,
26). Therefore, we designed two chimeric mutants by replacing either the N terminus (aa 1-205) and the C terminus, or only the C terminus (aa 290-336) of HOXA1, with the corresponding regions of HOXB1 (aa
1-174 and 258-296) (Fig.
10B), leaving the HOXA1
homeodomain and FDWM regions intact (Fig. 10B).
Expression vectors encoding the HOXA1/B1CT and the HOXB1/A1HD chimeric
proteins were cotransfected with PBX1 in P19 cells together with
reporters under the control of either the entire b1-ARE (pAdMLARE) or
R3 (pAdMLR3). In controls, both chimeras bind the b1-ARE R3 element
in vitro with comparable affinity, and both activate
transcription from the pAdMLR3 reporter in EC cells and COS-7 cells
(Fig. 10A and data not shown). Testing the response using
the entire b1-ARE control region, the transcriptional activity of the
chimera carrying only the C-terminal replacement remained low and
comparable to that of unmodified HOXA1. In contrast, the activity of
the chimera containing the combined C- and N-terminal replacement was
enhanced and comparable to that of wild type HOXB1 (Fig.
10A).
These results show that specific DNA recognition and cooperative
binding with PBX1, provided by the region encompassing the homeo- and
FDWM domains, are not responsible for the differential activity of
HOXA1 and HOXB1 on the b1-ARE. Conversely, the HOXB1 N-terminal region,
comprising the transcriptional activation domain, appears to contain
specific determinants that are able to respond to cues from the cell
background leading to a higher activity with respect to HOXA1.
Mammalian HOX proteins are a large family of transcription factors
that control cell identity, differentiation, and patterning in animal
embryonic development. Due to the similarities in their structure,
in vitro binding abilities, and in vivo function,
it is important to understand the factors and components that serve to
regulate the selectivity and specificity of HOX transcription complexes
during activation of downstream target genes. In this study we have
addressed some of these issues by examining the regulatory properties
and functional requirements of an in vivo Hox-responsive
target sequence represented by an auto-regulatory enhancer (b1-ARE)
from the Hoxb1 gene. We have used human or murine EC cells,
which retain most of the characteristics of primitive neuroectodermal
cells (32, 33), as a model system to test the overall transcriptional
activity of the b1-ARE. We found that the b1-ARE has both higher basal
and higher HOX/PBX-induced activities in neuro-ectodermal EC cells as
compared with other cell backgrounds. The enhanced EC activity
correlates with the presence of a bipartite motif in the b1-ARE that
binds SOX and OCT proteins and is necessary for the optimal response of
b1-ARE to transcriptional activation by HOX/PBX heterodimers both
in vitro and in vivo. Surprisingly, despite
recent evidence suggesting that Hox paralogs are functionally equivalent (57), our analysis revealed that there are differences in
the way that the b1-ARE responds to HOXB1 and HOXA1, which correlate
with differences in their N-terminal domains. Together, our results
have uncovered some novel aspects of how HOX proteins interact with
their in vivo target sequences, identified cis-elements that
can modulate HOX/PBX complex activities, and raised a number of
important issues on the functional differences between paralogous HOX proteins.
Cis-determinants in the b1-ARE--
Our analysis has revealed that
a highly conserved sequence motif located immediately upstream of a
HOX/PBX sequence (R3) is an important determinant of the cell
type-specific restriction of the b1-ARE activity. This bipartite
element mediates high affinity binding of a SOX/OCT heterodimer that
contributes to enhancer activity in addition to, and in combination
with, HOXA1/PBX1 and HOXB1/PBX1 heterodimers. This motif (SOct) allows
cooperative binding of SOX-2/OCT1 and SOX-2/OCT3/4 heterodimers present
in extracts of murine or human EC cells and mouse embryos. The binding of the SOX-2/OCT1 complex to the b1-ARE site actually appears to be
stronger than that observed with the SOX/OCT-binding sequence located
in the developmentally regulated FGF-4 enhancer (50, 51). This
difference could be explained by the different spacing between the SOX-
and OCT-specific hemisites on the two bipartite sequences. In the
b1-ARE sequence, the SOX and Octamer sites actually overlap by 1 base
pair, whereas in the FGF-4 enhancer the two sites are spaced apart by 2 base pairs. Since it was previously shown that the distance between the
two sites is critical for the assembly of the SOX/OCT heterodimer in
the context of the FGF-4 enhancer (46), it seems likely that the
configuration of the b1-ARE site allows a different interaction between
SOX-2 and OCT1 or OCT3/4, leading to a stronger binding of the
SOX-2/OCT complexes. The slightly different mobility of the
SOX-2/OCT3/4 complex on the b1-ARE versus the FGF-4 sequence
(see Fig. 5) might be caused by a differential bending of the DNA
induced by SOX-2 that might also affect binding strength. High affinity
binding of a SOX-2/OCT3/4 complex was recently reported on a closely
spaced bipartite sequence found in the upstream regulatory element of the murine UTF1 transcription factor (58).
The Hoxb1 SOX/OCT (SOct) site contains at its 5' end a
potential binding site (TGACAA) for the TALE homeodomain proteins MEIS and PREP. This is adjacent to R2 and separated from R3 by 17 nucleotides. A similar combination of a TALE site and a HOX/PBX site is
also found in an r4-restricted cross-regulatory enhancer from the
Hoxb2 gene, except that the sites are separated by 8 base
pairs (11, 15). In the case of Hoxb2 there is no overlapping
SOX/OCT site, but the TALE and HOX/PBX sites synergize in
vitro and in vivo to allow the formation of a trimeric
and transcriptionally active HOXB1-PBX1-MEIS/PREP complex required for
enhancer activity in transgenic assays (10, 11). In the context of the
b1-ARE, however, MEIS1 or PREP1 is not necessary for DNA binding of
HOX/PBX heterodimers and, accordingly, is not required to bind DNA
to enhance the transcriptional activity of the HOXB1/PBX1 complex in
transfected cells (8). Indeed, in EMSAs none of the retarded complexes
formed on the complete SOct sequence by either EC cell or mouse embryo
extracts appeared to contain MEIS or PREP proteins (data not shown).
The Role of OCT and SOX Proteins in b1-ARE Enhancer Activity in EC
Cells--
Transient transfection analysis showed that the SOct site
is responsible for the high basal transcriptional activity of the b1-ARE sequence in EC cells and is also required for fully activated transcription in the presence of HOXB1 and PBX1. Interestingly, transfection of SOX-2 and OCT1 in non-EC cells (COS-7), which do not
express endogenous SOX proteins, significantly increases both the basal
activity of the b1-ARE reporter and the HOXB1/PBX1-mediated transactivation. The nature of the OCT partner seems to be important, as transfection of SOX-2 and OCT3/4 had no effect on the b1-ARE. Conversely, a SOX-2/OCT3/4 complex but not a SOX-2/OCT1 complex was
reported to have a positive effect on the transcriptional activity of
the FGF-4 (51), osteopontin (47), and UTF1 (58) enhancers in HeLa
cells. These results indicate that the b1-ARE SOX/OCT site might have
unique binding properties in vivo compared with previously
reported bipartite sites and facilitates functional interaction
specifically for the SOX-2/OCT1 complex. Therefore, both sequence and
spacing between the two hemisites appear to be important general
factors in dictating specific binding of heterodimeric complexes
between SOX-2 and alternative OCT proteins to this class of target elements.
In Vivo Role for the SOX/OCT Site in b1-ARE Regulation--
These
experiments suggest a model whereby the bipartite SOX/OCT site
contributes to the activity of the b1-ARE enhancer in vivo
by recruiting SOX-2/OCT1 heterodimers. This in turn could either
increase the stability or the affinity of binding by HOXB1-PBX1 and
HOXA1/PBX1 complexes and/or activate transcription in synergy with such
complexes. In support of this, combined mutations in both parts of the
SOX/OCT site show that it is necessary for full in vivo
activity of the b1-ARE in transgenic mouse embryos.
Genetic and regulatory analyses have previously shown that
Hoxa1 and Hoxb1 function synergistically to
regulate the early r4 expression of Hoxb1 (7.5-8.5 dpc) and
that this segmental expression is maintained from 8.5 dpc and onwards
by Hoxb1 itself (6, 27, 28, 59). Mutation of both the SOX-
and OCT-specific hemisites in the b1-ARE does not abolish reporter
expression in r4 at 9.5 dpc (Fig. 8). This expression in later stages
implies that endogenous HOXB1/PBX complexes are still able to activate the mutant b1-ARE reporter. This is consistent with our findings in EC
cells that showed HOXB1/PBX complexes could stimulate transcription in
the absence of the SOct site, although not at maximal levels, whereas
the HOXA1/PBX complexes displayed an absolute requirement for this site
in transactivation (Fig. 4). In contrast, mutation of the SOct site
impairs the ability of the b1-ARE reporter to generate a stripe of
ectopic expression in r2 in response to in utero RA
treatment (Fig. 8). Furthermore, this ectopic RA response is also
abolished in Hoxa1 mutant embryos (Fig. 9). These results suggest that, as in EC cells, the interaction of HOXA1/PBX complexes with factors binding to the SOct site is essential for generating complexes with sufficient activity to trigger the b1-ARE
auto-regulatory loop in r2. This underscores the importance of the SOct
site for in vivo activity and differential dependence of
HOXA1/PBX versus HOXB1/PBX complexes upon this site for
modulating transcriptional activity. It is interesting that the ectopic
response of the transgene to RA depends upon HOXA1 and that HOXB1 does
not compensate. We have found that the reason for this is that while
both the Hoxa1 and Hoxb1 genes have 3'-RAREs
necessary for early neural expression, the Hoxb1 3'-RARE does not
respond to in utero RA treatment (data not shown).
A mutation in the OCT-specific hemisite, which is
recognized by the POU homeodomain of the Octamer factor, has little or
no effect in transgenic mice. This suggests that the SOX/OCT complex can assemble in vivo on a suboptimal target sequence,
through interaction of the SOX factor with the POU-specific domain of the Octamer factor. Similar data on the relative importance of the
POU-specific sequence for binding of OCT1 and OCT3/4 to a bipartite
SOX/OCT site in vitro was also observed in the case of the
UTF1 upstream regulatory element (58).
Selectivity and HOX Protein Determinants--
The homeodomains of
HOXB1 and HOXA1 are almost identical, and in combination with PBX
proteins display a virtually indistinguishable binding specificity.
Therefore, the differential activity of HOXB1 or HOXA1 complexes with
PBX most likely resides in specific interactions with other DNA-binding
factors and/or with the transcriptional machinery. Indeed, our analysis
demonstrates that a critical determinant of the difference between the
HOXB1/PBX1 complex and that of HOXA1/PBX1 resides in the HOXB1
N-terminal transcriptional activation domain. A chimeric protein, where
the N-terminal domain of HOXA1 is replaced with that of HOXB1, makes it
indistinguishable from wild type HOXB1 in activating the b1-ARE in
cooperation with PBX1. This suggests that DNA recognition is not the
key variable involved in modulating the activity of group-1 HOX
proteins on the b1-ARE target and that factors interacting with the N
terminus of these HOX proteins are responsible for mediating their
differential activities in a tissue- or cell-specific manner.
Although we have focused on how distinct HOX proteins in the
heterodimers differentially interact with cofactors, diversity in PBX
partners may also contribute to the selectivity. The existence of both
the PBX and other novel HOX cofactors might explain the observed
tissue-specific restriction of some Hox-responsive enhancers in vivo. A recent report (57) has challenged the view that
paralogous HOX proteins have gained intrinsic functional diversity in
the course of evolution. They propose that paralogous proteins are functionally equivalent, and it is only the relative levels or domains
of their expression that govern their unique activities. Our results,
conversely, show the existence of intrinsic differences in function
between the HOXA1 and HOXB1 proteins. In this regard, the difference
between HOXB1 and HOXA1 in their ability to interact functionally with
the same Hoxb1 auto-regulatory element in a specific cell
context is a clear example of non-redundancy between products of
Hox genes belonging to the same paralogous group. We feel
that this observation may also extend to other paralogous groups, and
it will be important to characterize the nature of the proteins
interacting with the N-terminal domains of the HOX proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
65 to +30).
pAdMLARE contains the AvaI-HaeII fragment of the
Hoxb1 r4-autoregulatory enhancer (b1-ARE) (6) cloned as a
PCR-amplified HindIII-XhoI fragment into pMLluc.
pAdMLR3 contains a trimer of repeat 3 of the b1-ARE (sequence,
5'-GATCCGGGGGGTGATGGATGGGCGCTGGGA-3') cloned as a
BamHI-HindIII fragment into pML. pAdMLARE
1-52,
R1, and
R1 + R2 have been obtained by cloning
PCR-amplified HindIII-XhoI fragments of the
b1-ARE (from nucleotides 53-140, 64-140, and 82-140, respectively)
into pMLluc. In pAdMLoctm, the Sox/Oct-binding sequence CTTTGTCATGCTAAT
in the b1-ARE was changed to GCAAGTCGACTGCCT. The pTUASluc reporter
construct was described (37).
-minimum Eagle's medium. Transfections were carried out by
CaPO4 precipitation (39). In a typical transfection experiment, 8 µg of reporter plasmid, 4-8 µg of expression
construct, and 0.2 µg of pCMV-
-gal (CLONTECH)
as an internal control were used per 10-cm dish. 48-60 h after
transfection, cells were washed and lysed directly on the plate with a
solution containing 1% Triton X-100, 25 mM glycyl-glycine,
pH 7.8, 15 mM MgSO4, 4 mM EGTA, 1 mM DTT. Extracts were collected, centrifuged to clear the
supernatant, and assayed for luciferase and
-galactosidase expression as described (40). To rule out the existence of intrinsic differences between COS-7 and P19 cells in sustaining transcriptional activation, we tested for the capability of a GAL4-VP16 chimera to
activate transcription from a GAL4-responding reported in both cell
lines. The GAL4-VP16 chimera gave identical results in terms of
transactivation in both cell lines (data not shown).
80 °C. To obtain
embryonic cell extracts, about 50 mouse embryos were collected at 9.5 dpc, washed with phosphate-buffered saline, and lysed as described
above. Gel retardation analysis was performed by preincubating the cell
extracts (8 µg) for 30 min on ice in 20 µl of binding buffer (75 mM NaCl, 20% Ficoll, 10 mM Tris-HCl, pH 7.5, 0.5 mM EDTA, 10 mM DTT, 3 µg of poly(dG-dC)
or poly(dI-dC)), together with 2 µl (0.5 ng, 5 × 104 cpm) of 32P-end-labeled oligonucleotide
probe. Competition experiments were carried out by adding 2 µl of
-Oct1 (Santa Cruz Biotechnology),
-Oct3/4 (kindly provided by
Hans Shöler), or
-Sox-2 (a gift from Marco E. Bianchi)
antisera before adding the probe. The incubation mixture was resolved
by electrophoresis on a 5% polyacrylamide gel in 0.25× TBE at 10 V/cm. Gels were dried and exposed to a Kodak X-AR film at
70 °C.
The b1-ARE oligonucleotide probe sequence was
5'-AGCTTGTGTCTTTGTCATGCTAATGATTGGGGGG-3'.
-galactosidase reporter activity in founder embryos was performed as
described previously (41). RA exposure was achieved by treating
pregnant females with embryos at 7.5 dpc by oral gavage with 200 ml of
sesame seed oil containing all-trans-retinoic acid (Sigma),
diluted from a 25 mg/ml stock solution in dimethyl sulfoxide, for a
final dose of about 20 mg/kg of maternal body weight (42). The
inductive response was assayed in embryos at 9.5 dpc.
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (27K):
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Fig. 1.
The HOXB1/PBX1 complex-mediated
transcriptional activation is significantly higher in neuroectodermal
versus other cell lines. Luciferase activity, in
arbitrary units, was assayed from extracts of the indicated transiently
transfected cell lines. The cells were transfected with 4 µg of the
SV40-driven HOXB1, and/or PBX1, expression constructs, together with 8 µg of alternatively pAdMLluc (C) or pAdMLARE. The
inset shows the low level luciferase activities in
control transfections. 0.2 µg of the pCMV -gal plasmid were
cotransfected in all experiments as an internal standard.
Bars represent the mean ± S.E. of at least four
independent experiments.
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Fig. 2.
Transcriptional activity mediated by b1-ARE
is mainly restricted to the HOXB1/PBX1 complex in neuroectodermal
cells. Luciferase activity, in arbitrary units, was assayed from
extracts of P19 cells. The cells were transfected with 4 µg of the
SV40-driven HOXA1, HOXB1, HOXB2, and/or PBX1 expression constructs
together with 8 µg of pAdMLluc (C), pAdMLARE
(ARE), or pAdMLR3 (R3). 0.2 µg of the
pCMV -gal plasmid were cotransfected in all experiments as an
internal standard. Bars represent the mean ± S.E. of
at least five independent experiments.
R1 + R2, Fig. 3A). Considering that a reporter containing a multimer of
the R3 motif alone responds equally well to HOXB1 or HOXA1 (Fig. 2), this result suggests that sequences flanking R3 are important in
mediating the selective preference of the b1-ARE for HOXB1.
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Fig. 3.
The b1-ARE selectivity is unaffected
by deletion of the R1 and R2 elements. A, Luciferase
activity, in arbitrary units, was assayed from extracts of P19 cells.
The cells were transfected with 4 µg of the SV40-driven HOXA1, HOXB1,
and/or PBX1 expression constructs, together with 8 µg of pAdMLluc
(C), pAdMLARE (ARE), or its deletion mutants
( 1-52,
R1, and
R1 + R2). 0.2 µg of the pCMV
-gal plasmid were cotransfected in all
experiments as an internal standard. B, schematic
representation of the Hoxb1-ARE and of its deletion mutant
derivatives. Numbers indicate the nucleotide
positions.
R1+R2+Sox, Fig.
4B) was made in the context of the smallest fragment
(ARE
R1+R2) showing differential response to
transactivation by HOXB1/PBX1 and HOXA1/PBX1 (see Fig. 3). This
mutation, like the ARE SOctm mutation, completely abolished the basal
activity of the reporter in P19 cells, reduced transactivation by the
HOXB1/PBX1 complex by 30%, and almost completely suppressed HOXA1/PBX
activity (Fig. 4A).
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Fig. 4.
The bipartite SOX/OCT-binding site is
necessary for basal and HOXB1/PBX1-induced activity of b1-ARE in P19
cells. A, luciferase activity, in arbitrary units, was
assayed from extracts of P19 cells. The cells were transfected with 4 µg of the SV40-driven expression constructs for HOXA1, HOXB1, and/or
for PBX1, together with 8 µg of pAdMLluc (C), pAdMLARE
(ARE), pAdMLR3 (R3), pAdMLSOctm
(SOctm), pAdMLARE R1+R2 (R1 + R2), or
pAdMLARE
R1 + R2 + SOX (
R1 + R2 + SOX). 0.2 µg of the pCMV
-gal plasmid were cotransfected in all experiments
as an internal standard. B, schematic representation of the
Hoxb1-ARE and of its mutant derivatives. Numbers
indicate the nucleotide positions.
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Fig. 5.
SOX/OCT1 and SOX/OCT3/4 complexes bind to the
b1-ARE in P19 and NT2/D1 cells. EMSA of double-stranded
oligonucleotides representing a region of 34 bp encompassing the SOct
site within b1-ARE (b1-ARE, left side) or the SOX/OCT
bipartite-binding site found within the FGF-4 promoter (FGF-4,
right side). Nuclear extracts from P19 or NT2/D1 cells were
challenged with the labeled oligonucleotides as described under
"Experimental Procedures." Anti-Oct1 and anti-Oct3/4-specific
antisera were used to characterize the two retarded complexes. An
asterisk indicates the position of a nonspecific retarded
complex found in NT2/D1 cells.
View larger version (65K):
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Fig. 6.
SOX/OCT1 and SOX/OCT3/4 complexes bind to the
b1-ARE in mouse E9.5 embryonic extracts. EMSA of double-stranded
oligonucleotides representing a region of 34 bp encompassing the SOct
site within b1-ARE (b1-ARE, left side) or the SOX/OCT
bipartite-binding site found within the FGF-4 promoter (FGF-4,
right side). Nuclear extracts from P19 or NT2/D1 cells and
whole cell extracts from embryonic day 9.5 (E.9.5) mouse
central nervous system were challenged with the labeled
oligonucleotides as described under "Experimental Procedures."
Anti-Oct1 and anti OCT3/4-specific antisera were used to characterize
the two retarded complexes. An asterisk indicates a
nonspecific binding complex in NT2/D1 cell extracts.
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Fig. 7.
SOX-2 activates transcription from the b1-ARE
in cooperation with OCT1 but not with OCT3/4 in COS-7 cells.
Results represent the fold activation over the b1-ARE basal luciferase
activity assayed from extracts of transiently transfected COS-7 cells.
The cells were transfected with 8 µg of pAdMLARE together with 4 µg
of the SV40-driven SOX-2, OCT1, OCT3/4, or HOXB1 and PBX1 expression
constructs as indicated. 0.2 µg of the pCMV -gal plasmid were
cotransfected in all experiments as an internal standard.
Bars represent the mean ± S.E. of at least four
independent experiments. An asterisk indicates a nonspecific
binding complex in NT2/D1 cell extracts.
G, and data not shown). However, there may be a quantitatively reduced level of activity in
this mutated b1-ARE element that is hard to detect, since
-galactosidase staining allows only a qualitative estimate of
transgene expression. These data show that a complete SOX/OCT site, and
presumably efficient binding of SOX/OCT heterodimer(s), in combination
with the HOX/PBC repeats are necessary in vivo for mediating
the RA response controlled by the conserved auto-regulatory elements in
the b1-ARE.
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Fig. 8.
Mutations in the SOX/OCT-binding site within
the Hoxb1 r4 enhancer (b1-ARE) influence the response
to RA but not r4-restricted expression. A-D, lateral
(A and C) and dorsal (B and
D) views of transgene expression in 9.5-10.0 dpc. embryos
carrying a lacZ reporter construct with a mutated form of
the OCT-specific hemisite (TAAT to CCGG) in the context of the
highly conserved 331-bp StuI-HindIII
Hoxb1 r4-autoregulatory enhancer (6). The embryo in
A and B was not exposed to ectopic RA, whereas
that in C and D was isolated from a female given
RA at 7.75 dpc by oral gavage. This mutation does not impair the
ability of the r4 enhancer to direct expression to r4 (A and
B) or to mediate a response to ectopic doses of RA as
indicated by the induction of a second stripe of expression in r2
(C and D). E-G, lateral (E
and F) and dorsal (G) views of reporter
expression in 9.0-10.5-dpc embryos treated with RA and carrying the
combined mutations in the SOX- (TTTGTC to CGCTGT) and OCT (TAAT to
CCGG)-specific hemisites. In both untreated and RA-treated cases r4
expression is not affected (E-G and data not shown).
However in the majority of cases (4/5) the double mutant construct
fails to mediate a response to ectopic RA (E), and in the
single example of an RA response the induction in r2 is very patchy and
incomplete (F and G), compared with wild type or
the single TAAT mutation (C and D). A schematic
diagram of the transgenic constructs and mutations is indicated
below the panels. The three bipartite HOX/PBX repeats are
indicated as R1-R3.
embryos show a robust r4
lacZ expression (Fig.
9A), whereas homozygous Hoxa1
/
untreated embryos show a
significantly reduced expression in r4 corresponding to the reduced r4
territory in these mutants (Fig. 9C). Upon treatment with
RA, 6/6 Hoxa1 wild type and 9/10 Hoxa1+/
heterozygous embryos responded by
ectopically expressing the transgene in the anterior hindbrain (Fig.
9B), whereas 5/5 homozygous Hoxa1 mutant embryos
failed to induce ectopically transgene expression (Fig.
9D).
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Fig. 9.
Targeted inactivation of Hoxa1
impairs the RA-induced ectopic activation of b1-ARE. Dorsal
(A-D) views of transgene expression in 9.5-10.0-dpc.
embryos carrying a Hoxb1 r4-autoregulatory enhancer-driven
lacZ reporter construct (6). A and B,
Hoxa1 wild type background. The embryo in A was
not exposed to ectopic RA, whereas that in B was isolated
from a female given RA at 7.75 dpc by oral gavage. The Hoxb1-ARE
lacZ reporter is ectopically activated by RA treatment and
displays a stripe of expression in r2 in 6/6 embryos (B).
C and D, Hoxa1 /
background. The
Hoxb1/lacZ transgene fails to be expressed
ectopically in r2 in 5/5 embryos isolated from females treated with RA
at 7.75 dpc by oral gavage (D). In both untreated
(C) and RA treated (D) cases r4 expression is not
affected. ov, otic vesicle.
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Fig. 10.
The determinants for the selective
transactivation of b1-ARE reside within the N-terminal transcriptional
activation domain of HOXB1. A, results represent the
fold activation over the b1-ARE basal luciferase activity assayed from
extracts of transiently transfected P19 cells. The cells were
transfected with 4 µg of the SV40-driven expression constructs for
HOXA1, HOXB1, or their chimeric derivatives, and/or for PBX1, together
with 8 µg of pAdMLluc (C), pAdMLARE
(ARE), or pAdMLR3 (R3). 0.2 µg of the pCMV -gal plasmid
were cotransfected in all experiments as an internal standard.
B, schematic representation of the HOXB1 and the HOXA1
proteins and of their chimeric derivatives. Numbers indicate
amino acid positions.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENT |
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We thank Hans Schoeler for the kind gift of anti-OCT3/4 antiserum.
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FOOTNOTES |
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* This work was supported by grants from the Telethon Foundation (to V. Z. and F. M.), the Italian Association for Cancer Research (to V. Z.), by an EEC Biotechnology Network Grant BIO4 CT-960378, and Medical Research Council (to R. K.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by an Human Science Frontier Program fellowship. Present address: Dept. of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, 240 Longwood Ave., Boston, MA 02115.
Supported by an EMBO postdoctoral fellowship.
** Supported by postdoctoral fellowships from EMBO and the Human Science Frontier Program.
¶¶ To whom correspondence should be addressed: DIBIT, Istituto Scientifico H.S. Raffaele, Via Olgettina, 58, 20132 Milano, Italy. Tel.: 39-02-26434805; Fax: 39-02-26434861; E-mail: zappavigna. vincenzo{at}hsr.it.
Published, JBC Papers in Press, March 1, 2001, DOI 10.1074/jbc.M011175200
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ABBREVIATIONS |
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The abbreviations used are: HD, homeodomain; r4, rhombomere 4; EC, embryonal carcinoma; RA, retinoic acid; RARE, RA response element; PCR, polymerase chain reaction; aa, amino acids; DTT, dithiothreitol; EMSA, electrophoretic mobility shift assay; dpc, days post-coitum; bp, base pair; FGF, fibroblast growth factor.
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