1 CNRS UMR 8542, Ecole Normale Supérieure, 46 rue d'Ulm, 75230 Paris
Cedex 05, France
2 Université Paris 7, UFR de Biologie, 2 place Jussieu, 75005 Paris,
France
* Author for correspondence (e-mail: prochian{at}wotan.ens.fr)
Accepted 24 January 2003
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SUMMARY |
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Key words: Cerebellum, Mes-metencephalon, Cytoskeleton, Homeodomain, Homeoprotein co-factors, Winged-helix/Forkhead box, Otx2, Goosecoid, Lim1
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INTRODUCTION |
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A probable explanation for homeoprotein specificity is their association
with cofactors. Clearly, homeoproteins have shown associations with numerous
proteins, including members of the same homeoprotein family, members of
different homeoproteins and non-homeodomain proteins. For example, association
of Hox and Engrailed with homeoproteins of the PBC class (Drosophila
Exd and its vertebrate homologs Pbx1, 2, 3) enhances their DNA-binding
specificity and/or affinity (reviewed by
Mann and Chan, 1996). In fact,
many homeoprotein molecular partners have recently been identified. They
belong to several classes of transcription regulators Groucho
(Jimenez et al., 1997
), Smad
(Germain et al., 2000
;
Shi et al., 1999
), GATA4
(Lee et al., 1998
), Nuclear
hormone receptor (Budhram-Mahadeo et al.,
1998
; Kakizawa et al.,
1999
), bHLH (Poulin et al.,
2000
), CREB (Edelman et al.,
2000
), SRF (Carson et al.,
2000
) and Maf (Kataoka et al.,
2001
).
The possibility that homeoproteins also interact with the
winged-helix/Forkhead box transcription factor HNF3ß/Foxa2 (called Foxa2
in the new nomenclature) first came from mice null for Foxa2 and Goosecoid
(Gsc) or Lim1. Indeed, the phenotype of these mice suggested that the two
latter factors genetically interact with Foxa2 early in development
(Filosa et al., 1997;
Perea-Gomez et al., 1999
). In
these two cases, however, direct protein-protein interactions were not
investigated. More recently, Foxa2 has been shown to interact directly with
homeoprotein Otx2, hence repressing Otx2-directed gene expression ex vivo
(Nakano et al., 2000
). Another
report documents how direct binding of Foxa2 to Pdx1 mediates cooperative
interactions of the complex with an enhancer element of Pdx1 and
regulates Pdx1 expression in pancreatic ß-cells
(Marshak et al., 2000
).
Recently, we demonstrated that Hoxa5 binds Foxa2 and FKHR and that this
binding bears important transcriptional and physiological consequences, in
particular in the control of body growth
(Foucher et al., 2002
).
Finally FKHR also interacts with Hoxa10 in endometrial cells of the uterus
(Kim et al., 2003
).
Foxa2 is expressed in different regions of the developing nervous system.
In the ventral mesencephalon and cerebellum Foxa2 is co-expressed with
Engrailed homeoproteins En1 and En2, from now on collectively called
Engrailed (Dahmane and Ruiz-i-Altaba,
1999; Davis and Joyner,
1988
; Hynes et al.,
1995
; Sasaki and Hogan,
1994
) raising the possibility that the two transcription factors
interact to regulate common target genes. In a previous report, it was found
that the neuronal Microtubule-associated protein 1B
(MAP1B/Mtap1b) promoter is regulated by Engrailed and a region of the
promoter, conserved between man and rodent, primarily responsible for this
regulation, was identified (Montesinos et
al., 2001
). In the present work, we show that this promoter
fragment contains two conserved overlapping binding sites for Foxa2 and
homeoproteins and, when incubated with cerebellum nuclear extracts from the
newborn mouse, associates with a protein complex that includes Engrailed and
Foxa2. Because of the importance of the interaction between Forkhead box and
homeodomain transcription factors we have taken Engrailed and Foxa2 as
archetypes of the families, analyzed their molecular and physiological
interactions, and identified the domains engaged in their interaction.
Interacting regions have been further analyzed for other homeoprotein-Foxa2
interactions, leading to the identification of highly conserved domains for
both families of proteins. This strongly suggests that the co-regulation of
common targets by Forkhead box transcription factors and homeodomain proteins
is a general phenomenon.
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MATERIALS AND METHODS |
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Plasmids and oligonucleotides
N- and C-terminal deletions of Foxa2, Hoxa5, Lim1 and Gsc were generated by
PCR using Pfu polymerase (Promega). T7 promoter was added directly through
incorporation into the 5' oligonucleotide sequence. Owing to low yields
of amplification, Hoxa5, Lim1 and Gsc fragments were sub-cloned in pBluescript
SKII (Stratagene). The pCMV-Foxa2 expression plasmids [a gift from Dr P.
Steenbergh and Dr G. R. Crabtree (Pani et
al., 1992)] was used as the PCR-template. GST-En2 (a gift from Dr
A. Joliot, ENS, Paris) and GST-Foxa2 fusions were prepared by inserting chick
En2 and rat Foxa2 coding sequences into a modified form of pGEX1 (Amersham
Pharmacia Biotech). pGEXEn2 was subsequently used to create the Gsc and Lim1
GST fusion proteins. The latter open reading frames were amplified by PCR
using Pfu with primers containing appropriate restriction sites in the
primers. Truncated constructs of En2 fused to GST, generated by PCR, were a
gift from Dr A. Maizel (ENS, Paris). pCLHA-Foxa2 was constructed by first
inserting a SacI site in a modified version of pCMV-Foxa2 and then
swapping the Foxa2 open reading frame (SacI-EcoRI fragment)
with Hoxa5 in pCLHAHoxa5. Plasmids containing whole or parts of the
MAP1B promoter have been described previously
(Montesinos et al., 2001
).
pMAP
HF1+2-luc was constructed by inverse PCR reactions using
the ExSite mutagenesis kit (Stratagene).
Recombinant protein production
GST-En2, GST-Foxa2, GST-Hoxa5, GST-Gsc, GST-Lim1 and GST proteins were
produced in E. coli BL21 strain and purified on glutathione-Sepharose
4B beads (Amersham Pharmacia Biotech), according to manufacturer's
instructions. En2SP, a deleted version of En2 (amino acids 1 to 9
followed by amino acids 186-289), was produced from expression plasmid
pTrc9mEn2
SP (Montesinos et al.,
2001
) and purified on Hitrap heparin-Sepharose columns (Amersham
Pharmacia Biotech). Protein concentration was determined by a modified
Bradford assay (Bio-Rad) using bovine serum albumin (BSA) as a standard. All
samples were verified by SDS-PAGE.
35S-labeled En2, Hoxa5, Gsc, Lim1 and Krox20 were produced using
the TNT Quick Coupled Transcription/Translation system or TNT T7 Quick for PCR
DNA (Promega), using pKEn2, pKHX13A
(Chatelin et al., 1996), pKSGsc
(a gift from Dr M. Schaeffer, Karlsruhe), pKSLim1 (a gift from Dr S.-L. Ang,
Strasbourg), and pETKrox20 (a gift from Dr P. Charnay, Paris) as templates.
35S-labeled fragments of these proteins were produced in the same
way, using PCR-amplified DNA fragments obtained as described above.
Pull-down interaction assay
Binding assays were performed in a final volume of 200 µl of binding
buffer (BF1: 20 mM Tris HCl pH 7, 100 mM NaCl, 1 mM EDTA, 10% glycerol, 0.01%
Nonidet P-40) by incubating 100 ng of glutathione-immobilized fusion proteins
with 1 µl of 35S-labeled Foxa2, Engrailed, Hoxa5, Gsc or Lim1
variants. Beads were rinsed with 3 ml of BF1-100 mM NaCl and 1 ml of BF1-500
mM NaCl, boiled for 5 minutes before analysis by SDS-PAGE and autoradiography.
In the case of GST-En2 mutants, 1 µg of each mutant was used.
Nuclear extracts and electromobility shift assays (EMSA)
Nuclear extracts from mouse neonatal (P0) cerebellum and posterior
mesencephalon were prepared as described previously
(Beckmann et al., 1997).
Dissected tissues were homogenized in 20 mM Hepes pH 7.9, 100 mM NaCl, 1.5 mM
MgCl2, 0.5 mM EDTA, 0.7% Nonidet P-40, 0.5 mM dithiothreitol, 10%
(wt/vol) glycerol and protease inhibitor cocktail Complete 1x (Roche
Diagnostics). After centrifugation (10 minutes, 2,000 g) and
washes in the same buffer, pellets were resuspended in 20 mM Hepes pH 7.9, 0.5
M KCl, 0.5 mM EDTA, 0.5 mM dithiothreitol, 25% (wt/vol) glycerol and Complete
1x and then incubated for 30 minutes at 4°C on a rocker. Nuclear debris
were removed by centrifugation at 15,000 g for 30 minutes at
4°C. Protein concentration was determined as for recombinant proteins.
DNA fragments (C, D, E) and oligonucleotides (HF1 upper strand:
5'-GCATATTAAGAAAAGAAATCTGTATC-3' and HF2 upper strand:
5'-GTATCTAGCATAATATGTCTGCC-3') were end-labeled by filling with
Klenow-fragment polymerase and [-32P]dCTP (Amersham).
Binding reactions were performed in a final volume of 20 µl (15 mM Hepes pH
8.0, 0.5 mM dithiothreitol, 6.25 mM MgCl2, 12.5% glycerol, 1 µg
of salmon sperm DNA and 10 µg BSA). Salt concentrations and/or glycerol
varied: 30 mM NaCl (Fig. 2A), 80 mM KCl, 25% glycerol (Fig.
2B), 90 mM NaCl (Fig.
4A,B). After incubation on ice for 30 minutes with 0.5 ng of each
probe, DNA-protein complexes were analyzed on 4% polyacrylamide gels
(acrylamide:bisacrylamide, 60:1) in 0.25x TBE buffer and 2.5% glycerol.
For supershift experiments, probes were first incubated with 1 µg of
nuclear extracts for 30 minutes on ice, and then for an additional 30 minutes
with 0.8 µl Foxa2 monoclonal antibody (clone 4C7, DSHB, Iowa City). In some
control experiments, another unrelated monoclonal antibody [9E10, anti-myc
(Evan et al., 1985
)] was also
used. For oligonucleotide binding, no BSA was included and only 100 ng of
salmon sperm DNA were used. In some experiments, En2
SP was
pre-incubated with GST-Foxa2 for 20 minutes before probe addition for a
further 30 minutes. When indicated, En2
SP was pre-incubated with the
probes (20 minutes) before addition of GST-Foxa2 (30 minutes). Gels were
pre-run at 4°C for 45 minutes at 130 V and run at 4°C for 1.5 hours at
240 V, dried and subjected to autoradiography.
|
|
The pCLHA-Foxa2 and a CMV promoter-driven myc-tagged Chick En2 plasmid
[pCL9mEn2 (Mainguy et al.,
2000)] were electroporated using a Bio-Rad Gene Pulser II
apparatus and 4-mm gap cuvettes (170 V and 950 µF in 250 µl culture
medium). One million cells were transfected with 2 µg of reporter plasmid
and the indicated amounts of expression plasmid, plus appropriate empty vector
to keep total DNA constant (12 µg). Transfection rate and protein nuclear
localization were determined by immunocytochemistry with anti-myc 9E10
(Evan et al., 1985
) and
anti-HA (3F10, Roche Diagnostics) antibodies. Cell viability after
transfection was checked using the MTT method
(Liu et al., 1997
).
Neuronal primary cultures were prepared as described previously
(Montesinos et al., 2001).
Mes-metencephalic regions from 13.5 d.p.c. mouse embryos were dissected and
dissociated, and cells were plated at a density of 250,000 cells/well in
24-well dishes. Transfections of neurons with 1 µg of reporter plasmid were
performed using the LipofectAMINE 2000 Reagent (Invitrogen) following the
manufacturer's instructions.
Luciferase activity was measured 24 hours after transfection
(Le Roux et al., 1995) in a
Lumat luminometer (Berthold). Results presented are the mean of three
independent experiments.
Quantitative RT-PCR
Total RNA from N2A and primary cultures of mes-metencephalic E13.5 mouse
nerve cells was isolated using the RNeasy kit and DNase-treated on column with
the Rnase-Free DNase Set (Qiagen). First-strand cDNA was synthesized from 450
ng of total RNA, using the Superscript II (Invitrogen) reverse transcriptase
and oligo(dT) following the supplier's protocol. Genomic DNA contamination was
systematically checked in samples without reverse transcriptase.
Real-time PCR was performed in a LigthCycler apparatus (Roche), using the LightCycler-FastStart DNA Master SYBR Green I kit (Roche). Diluted samples of cDNA derived from 2, 5, 10 or 20 ng of total RNA were used as template. Oligonucleotides used to amplify mouse En1, En2, Foxa2 and GAPDH sequences were: mEn1-fw: 5'-TGTGTTTCCTTGTGTGTCTGC; mEn1-rv: 5'-GTCTCCAGAAAAGGAAGGGG; mEn2-fw: 5'-AACAAGCGGGCCAAAATCAAGAA; mEn2-rv: 5'-CGCCCTGCTCGCCCTACTC; mFoxa2-fw: 5'-CACAGCCACCACCACCATCAG; mFoxa2-rv: 5'-GCATCCAGGCTCGCTTTGTTC; GAPDH-fw: 5'-TGACGTGCCGCCTGGAGAAAC; GAPDH-rv: 5'-CCGGCATCGAAGGTGGAAGAGT. The PCR program consisted in an initial step of 8 minutes at 95°C for polymerase activation, and 40 cycles as follows: 15 seconds at 95°C; 5 seconds at 60°C; 15 seconds at 72°C. Melting analysis of PCR products was performed to verify the specificity of the amplification reaction. Amplification efficiencies of targets in the conditions described were close to 100%.
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RESULTS |
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A short conserved region of the MAP1B promoter containing two
homeoprotein/Forkhead box binding sites has a regulatory function in
mesmetencephalic neurons
Interestingly, within fragment C, two regions contain closely associated
Foxa2 and homeoprotein cognate binding sites. The first Foxa2 binding site is
fused to an ATTA/TAAT site at its 5' extremity (defined as HF1, for
homeoprotein/Fox 1) and the second contains an internal TAAT/ATTA sequence,
defined as HF2 (Figs 1,
4). The functional properties
and physiological relevance of these potential targets of both Foxa2 and the
homeoproteins were further investigated. To that end, we prepared a truncated
version of the pMAP1B promoter (pMAPHF1+2-luc), in
which a 43 bp fragment including the HF1 and HF2 sites was excised. Expression
of the wild-type and deleted promoters fused to a luciferase reporter was then
analyzed in transfected primary cultures of mes-metencephalic E13 mouse
neurons expressing both Engrailed and Foxa2
(Fig. 3B). As shown in
Fig. 3A, the expression of the
deleted MAP1B promoter (pMAP
HF1+2-luc) is threefold
that of the wild-type promoter, demonstrating that the 43 bp fragment
including sites HF1 and HF2 has a regulatory function in mes-metencephalic
neurons.
|
Taken together these results suggest that Foxa2 and Engrailed physically interact and/or compete for overlapping DNA target sequences. The HF1 and HF2 sites described above have regulatory functions in mes-metencephalic neurons, and therefore the binding ability of Foxa2 and Engrailed to HF1 and HF2 sequences, alone or together, was investigated.
Foxa2 inhibits the binding of Engrailed to HF1 and HF2 sequences
The binding of GST-Foxa2 and/or En2SP (a shorter version of
Engrailed, see Materials and Methods) to synthetic oligonucleotides containing
HF1 shows that En2
SP binds HF1 and forms either one or two retarded
bands depending on its concentration (Fig.
4A, left panel). GST-Foxa2 binding to HF1 is weak and is only
visualized after long exposure times (Fig.
4A, middle panel). Fig.
4A (right panel) illustrates that GST-Foxa2 displaces En2
SP
from HF1 in a dose-dependent manner (compare lane 3 with lanes 8 and 9). This
decrease in En2
SP binding is concomitant with a slight increase in
GST-Foxa2 binding (compare lane 9 with lane 7), suggesting that En2
SP
favors the binding of Foxa2 to HF1.
En2SP also binds HF2, generating one shifted band
(Fig. 4B lanes 2-3), but
GST-Foxa2 binding could not be demonstrated
(Fig. 4B, lanes 4 and 5), even
after long exposures. Despite its apparent absence of binding to HF2,
GST-Foxa2 inhibited that of En2
SP even when En2
SP was
pre-incubated with HF2 for 20 minutes before GST-Foxa2 addition
(Fig. 4B, compare lane 2 with
lanes 6 and 7, and lane 3 with lanes 8 and 9). This latter experiment strongly
suggests that GST-Foxa2 binds En2
SP and that, as a result, the
En2
SP protein detaches from HF2. This prompted us to search for direct
physical interactions between Engrailed and Foxa2.
En2 directly binds Foxa2: identification of interacting domains in
En2 and Foxa2
GST-En2 was incubated with radioactive Foxa2 generated by in vitro
transcription and translation. As shown in
Fig. 5A, 35S-labeled
Foxa2 binds GST-En2. This interaction is specific since it is not observed
between 35S-labeled Foxa2 and GST alone
(Fig. 5A) or
35S-labeled luciferase and GST-En2 (not shown). To identify the
domains of Foxa2 that interact with Engrailed, 35S-labeled
truncated versions of Foxa2 (Fig.
5B) were generated and incubated with GST-En2. Oligonucleotide
primers were chosen to specifically delete Foxa2-characterized domains CRI to
CRIV (Wang et al., 2000)
(Fig. 5B). The pull-down
experiments (see Fig. 5C)
demonstrate that the CRI DNA-binding Forkhead domain (amino acids 148-257) is
necessary and sufficient for binding to GST-En2.
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Finally, to identify which domains of the homeoproteins Hoxa5, Gsc and Lim1
are involved in the interaction with Foxa2, radioactive fragments of these
homeoproteins, including known domains (i.e. the homeodomain and the
hexapeptide motif), were synthesized and tested for binding to GST-Foxa2 (not
shown). The results of these pull-down experiments are summarized in
Fig. 8 which also includes the
data reported by Nakano et al. (Nakano et
al., 2000) on Foxa2/Otx2 interacting domains. From these mapping
experiments performed on five distinct homoproteins, Engrailed, Hoxa5, Gsc,
Lim1 (this paper) and Otx2 (Nakano et al.,
2000
), it appears that in addition to the homeodomain, which is
sufficient for binding to Foxa2, other interacting domains can exist in either
the N-terminal (Engrailed, Hoxa5, Gsc) or C-terminal (Otx2) domains of
homeoproteins.
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DISCUSSION |
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A physical interaction between Foxa2 and Engrailed and its
physiological relevance
The existence of interactions between Foxa2 and Engrailed is supported by
the following observations. First, in fragment C conserved consensus ATTA/TAAT
homeoprotein binding sites are present in the vicinity of Foxa2 binding sites.
Second, within nuclear extracts, Engrailed binds the MAP1B promoter
in a complex that includes Foxa2 (this study)
(Montesinos et al., 2001).
Third, gel mobility-shift data obtained with HF2 sites are best interpreted in
terms of protein-protein interaction. Indeed, although Foxa2 does not show any
detectable binding activity to the HF2 site, it displaces En2
SP from
this DNA sequence. The absence of Foxa2 binding precludes a competition
mechanism and therefore favors a model in which Foxa2 binds En2
SP, and
provokes its dissociation from DNA. Similar inhibitory interactions have been
described for En1/Pax6, Ey/Antp, Hox/Maf and CDP/SATB1
(Jinqi et al., 1999
;
Kataoka et al., 2001
;
Plaza et al., 1997
;
Plaza et al., 2001
). In this
study, the interaction between the two transcription factors results in the
displacement of Engrailed from a cognate binding site. This was also observed
for Otx2 (Nakano et al., 2000
)
but is not a general rule since the two partners can show cooperative binding
to specific enhancer elements [Pdx1
(Marshak et al., 2000
)].
Finally, as will be discussed later, the proteins interact directly and the
interaction domains have been identified.
Two main factors indicate a physiological interplay of Engrailed and Foxa2
in regulating MAP1B expression. First, deleting the 43 bp fragment
encompassing HF1 and HF2 binding sites up-regulates MAP1B promoter
activity in mesmetencephalic neurons expressing Engrailed and Foxa2. Second,
in a cell context devoid of Engrailed or Foxa2 (N2A), dose-dependent
gain-of-function experiments demonstrate a regulatory activity of both
transcription factors as well as an interaction between the two factors to
regulate MAP1B promoter activity. In the latter experiment each
transcription factor acts as a co-factor for the other one. Separately the two
factors activate MAP1B at high expression levels and have no effect
at low levels but, in co-expression experiments, low levels of either factor
down-regulate the activity of the other one. This pattern of regulation
suggests at least two possible and non exclusive modes of interaction: binding
of the first factor conferring access to the second through conformational
changes of the promoter (Fig.
4A) or a direct interaction between the two factors
(Fig. 4B). In fact it is well
accepted that, depending on the cellular context (i.e. co-expression of
co-factors), some transcription factors have opposite functions on a given
promoter. For example, Engrailed has opposite regulatory functions on the
polyhomeotic promoter in Drosophila, depending on both
Engrailed concentration and Extradenticle expression
(Serrano and Maschat,
1998).
Foxa2 and homeoproteins interact through their conserved DNA-binding
sequences with various additional interacting domains in homeoproteins
Mapping of the interacting domains identified the Forkhead box binding
domain in Foxa2 as the only domain interacting with Engrailed, Hoxa5, Lim1,
and Gsc (this study) and Otx2 (Nakano et
al., 2000). Similarly, for all homeoproteins tested, the
homeodomain alone binds Foxa2. However, and in contrast with Foxa2, four out
of these five homeoproteins contained additional Foxa2-interacting regions:
Engrailed, Hoxa5, Gsc (in all three cases in the N-terminal sequence; this
study) and Otx2 [in its C-terminal sequence
(Nakano et al., 2000
)]. In
this study a detailed analysis of the interacting domains has been done for
Engrailed only and the mapping of the other homeoproteins has been limited to
the homeodomain, and its flanking N- and C-terminal regions, at large. In the
case of Engrailed, in addition to the homeodomain, a short sequence (amino
acids 146-199) overlapping the Pbx-interacting domain also binds Foxa2. This
latter domain and the homeodomain bind independently to Foxa2 and the
possibility that they interact with different sub-regions of the Forkhead box
domain was not investigated. Such an additional non-homeodomain Foxa2
interacting domain was also present in the N-terminal sequences of Hoxa5 and
Gsc, but not in Lim1. With the exception of the hexapeptide sequence present
in Engrailed and Hoxa5 (see below), no further similarities were found between
the Foxa2-binding domains identified outside the homeodomain in Engrailed,
Hoxa5, Gsc and Otx2. It is thus possible that, in addition to the homeodomain,
different homeoproteins have evolved separate Foxa2-binding regions with
regulatory functions.
In this context it is interesting that the fragment 146-199 of Engrailed
includes the EH2 (homologous to hexapeptide in Hox proteins) and EH3 domains
of Engrailed both implicated in functional interactions with Exd/Pbx
homeoproteins (Peltenburg and Murre,
1996). The same observation also stands for Hoxa5, for which the
N-terminal sequence containing the hexapeptide sequence binds Foxa2. A
possibility, not investigated here, is that both Pbx and Foxa2 bind Engrailed
(or Hoxa5) to form a tripartite complex or, alternatively, that Foxa2 and Pbx
binding are mutually exclusive. Also intriguing is the fact that Engrailed and
Gsc, as well as different Forkhead box proteins including BF1 and
Foxa2 interact with co-factors of the Groucho/TLE family
(Chen and Courey, 2000
;
Wang et al., 2000
;
Yao et al., 2001
). Since the
Groucho/TLE-interacting domains of Engrailed and Foxa2 have been mapped to the
EH1 and CRII domains, respectively (two domains not involved in the
Foxa2-Engrailed interaction) it is possible that larger complexes involving
Groucho/TLE proteins, Forkhead transcription factors and homeoproteins form in
vivo.
How general is the interaction between homeoproteins and Forkhead box
transcription factors?
The Forkhead box DNA-binding domain and the homeodomain are highly
conserved among winged-helix/Forkhead box transcription factors and
homeoproteins, respectively. This observation lends weight to the idea that
interactions between Fox proteins and homeoproteins could be a general
phenomenon. This is supported by the report of direct physical interactions
between Foxa2 and Otx2 (Nakano et al.,
2000) or Pdx1 (Marshak et al.,
2000
). In addition, Foxa2 interacts genetically with
Lim1 (Filosa et al.,
1997
) and Gsc
(Perea-Gomez et al., 1999
).
The former interaction regulates Sonic hedgehog expression in the
neural tube and the latter participates in the organizer activity of the
visceral endoderm. This led us to investigate if (and to demonstrate that)
Foxa2 binds Lim1 and Gsc. Homeoproteins that bind Foxa2 therefore presently
include Otx2, Pdx1, Hoxa5, Engrailed, Lim1 and Gsc, and the conservation of
some of the binding sequences suggests that this is probably a general rule
for both classes of partners, the homeoproteins (see above), but also the
Forkhead box transcription factors. In line with this hypothesis, we recently
showed that such interactions are not limited to Foxa2, but that they also
exist for another Forkhead box transcription factor playing a key role in
regulating IGFBP1 (Igfbp1) expression, FKHR (Foxo1). Indeed,
Hoxa5 physically interacts with FKHR, and this interaction has important
physiological consequences in regulating the IGFBP1 promoter in the
liver, a key parameter in postnatal growth
(Foucher et al., 2002
). A
similar physical interaction between FKHR and Hoxa10 was also reported, and it
was shown that both transcription factors cooperate on FKHR-binding sites,
within the IGFBP1 promoter, to regulate its cyclic activity in cells
of the adult uterus (Kim et al.,
2003
). Taken together, these data demonstrate that Fox proteins
and homeoprotein can interact physically and functionally to regulate many
distinct functions, from the earliest events of embryonic development
throughout adulthood.
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ACKNOWLEDGMENTS |
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