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First published online 26 March 2008
doi: 10.1242/dev.018028
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1 Biozentrum, University of Basel, Klingelberstrasse 70, CH-4056 Basel,
Switzerland.
2 ETH Zurich, Department of Biosystems, CH-4058 Basel, Switzerland.
3 CNS-Centre de Biologie du Developpement, 118 route de NARBONNE, Bat 4R3, 31062
Toulouse, France.
4 Facultad de Ciencas Biologicas UANL, Cuidad Universitaria, C.P. 66450,
Mexico.
5 Micromet AG, Am Klopferspitz 19, 82152 Martinsried/Munich, Germany.
* Author for correspondence (e-mail: walter.gehring{at}unibas.ch)
Accepted 1 March 2008
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: ANTP, HOX, bip2, dTAF3, Drosophila, Eye-to-wing transformation, Homeotic transformation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Wakimoto and Kaufman, 1981;
Lin and McGinnis, 1992;
Akam, 1998;
Mann and Morata, 2000). Upon
ectopic expression (gain-of-function) or loss-of-function mutations in HOX
genes, massive morphological changes are induced imposing a new segmental
identity, transforming parts or complete segments into another one
(Lewis, 1978). This finding
illustrates that a single protein regulates many cellular fates in one or more
segments. HOX genes encode highly conserved transcription factors that share a
common sequence element of 180 bp, the homeobox, which encodes a 60 amino acid
homeodomain (HD) that represents the DNA-binding domain and allows
sequence-specific recognition within the regulatory region of its target genes
(McGinnis et al., 1984;
Scott and Weiner, 1984).
However, the HD exerts a relatively low DNA-binding specificity
(Ekker et al., 1994;
Gehring et al., 1994). In
general, the third 
-helix of the HD binds a 6-bp DNA sequence
containing a TAATC/GC/G recognition core
(Ekker et al., 1991). This
recognition sequence appears statistically once per kilobase in the genome,
raising the question of how HOX proteins recognize their real target sequences
among other potential target sites to achieve segmental specificity. Studies
using chimeric HOX proteins indicate that the N-terminal arm of the HD that
contacts the adjacent minor groove of the DNA is in some cases sufficient to
provide specificity (Furukubo-Tokunaga et
al., 1993; Zeng et al.,
1993; Passner et al.,
1999; Berry and Gehring,
2000). However, sequences outside the HD were also found to be
important in conferring specificity (Lin
and McGinnis, 1992; Chan and
Mann, 1993; Zeng et al.,
1993; Chauvet et al.,
2000; Gebelein et al.,
2002; Merabet et al.,
2003). More recent experiments suggest that the homeodomain
recognizes the DNA structure in the minor groove rather than reading a
specific DNA sequence directly (Joshi et
al., 2007).
Two models for HOX gene specificity, the `widespread binding' and the
`co-selective binding' models have been proposed. The first one assumes
co-operative binding on multiple monomer-binding sites, increasing the
presence of one HOX protein on a cis-regulatory element, allowing the
regulation of the downstream target genes
(Biggin and McGinnis,
1997).
The second model proposes the regulation of target genes through protein
co-factors that increase the DNA-binding selectivity and affinity
(Biggin and McGinnis, 1997).
One factor contributing to HOX specificity was shown to be Extradenticle
(EXD). Mutations in exd lead to homeotic transformations without
affecting the expression pattern of the HOX genes
(Gonzalez-Crespo and Morata,
1995; Peifer and Wieschaus,
1990). EXD was consequently shown to act as a HOX co-factor
increasing the DNA-binding specificity and target site selectivity of HOX
proteins (Mann and Chan,
1996). The interaction of the HOX proteins with EXD involves a
highly conserved peptide motif, the YPWM motif, which contacts the HD of EXD,
as shown by structural analysis (Passner
et al., 1999; Piper et al.,
1999). The YPWM motif, shown to serve as a protein-protein
interaction motif, is highly conserved throughout the animal kingdom and lies
amino terminally to the HD. All HOX proteins share the YPWM motif except for
the Abdominal B (ABD-B) class of HOX genes, which have retained only a remnant
tryptophan at the corresponding position
(Izpisua-Belmonte et al.,
1991).
Interestingly, genetic experiments indicate that some HOX gene functions
and target genes are controlled independently of exd
(Peifer and Wieschaus, 1990;
Percival-Smith and Hayden,
1998), and removal of the YPWM motif does not completely abolish
EXD-HOX binding interactions (Galant et
al., 2002; Merabet et al.,
2003). The YPWM apparently serves other functions besides binding
EXD (Chan and Mann, 1996;
Merabet et al., 2003),
suggesting that other YPWM-motif-interacting co-factors might be involved.
Based on these findings, work on the HOX gene abdominal A
(abd-A) revealed a function of the YPWM motif in transcriptional
activation rather than DNA-binding selectivity
(Merabet et al., 2003). The
HOX gene Antennapedia (Antp) specifies the second thoracic
segment (T2) with a pair of wings and a pair of middle legs in D.
melanogaster. When ectopically expressed, Antp transforms head
structures into parts of the second thoracic segment, such as the antenna into
a middle leg and the dorsal head capsule into notum structures
(Schneuwly et al., 1987).
Antp further inhibits eye development by inducing cells co-expressing
eyeless (ey) and Antp to undergo apoptosis
(Plaza et al., 2001). In
combination with a constitutively active form of the Notch receptor
(Nact), which prevents cells from undergoing apoptosis,
Antp is able to transform the dorsal part of the eye into the
corresponding dorsal T2 appendage, the wing
(Kurata et al., 2000). Using
the OK-107 driver, ANTP is also capable of transforming the eye into wing
structures without Nact (see Results).
On the basis of these results, we have analyzed the role of the YPWM motif
and the DNA-binding specificity of Antp in inducing antenna-to-leg
and eye-to-wing transformations. We found that the YPWM motif and the
DNA-binding specificity of the HD are absolutely required for eye-to-wing
transformations. By contrast, the transformation of the antenna into a T2 leg
is largely dependent on the DNA-binding specificity, and to a much lesser
extent on the YPWM motif. Based on the strict requirement of the YPWM motif to
transform an eye into a wing, we screened for YPWM-motif-specific interacting
co-factors. Employing the yeast two-hybrid system, we identified a novel
Antp co-factor bric-à-brac interacting protein 2
(bip2), also referred to as dTAFII3 or TAFII155
(Gangloff et al., 2001), which
specifically interacts with the YPWM motif of Antp in vitro and in
vivo. Using gain- and loss-of-function approaches, we show that bip2
genetically interacts with Antp promoting dorsal ectopic wing
development
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Flies were reared on standard medium at 25°C. Lines used were:
ey-Gal4 (Halder et al.,
1998; Hauck et al.,
1999); OK-107-Gal4 (Connolly
et al., 1996); dpp-Gal4
(Staehling-Hampton et al.,
1994); UAS-Antp and UAS-AntpdHD
(Bello et al., 1998);
UAS-AntpQ50K (Plaza et
al., 2001); UAS-Nact
(Fortini et al., 1993);
UAS-Ubx, UAS-AntpAAAA, UAS-Bip2,
UAS-Bip2-HA, UAS-exd, UAS-GFP and wg-lacZ
(Couso et al., 1994);
eyg-lacZ (Jang et al.,
2003); Su(H)-lacZ
(Furriols and Bray, 2001); and
dpp-lacZ (Halder et al.,
1998).
Antibody staining
Staged larvae were dissected in cold PBS and fixed in PEM [100 mM Pipes (pH
6.9), 2 mM MgSO4, 1 mM EGTA, 4% formaldehyde] for 25 minutes on
ice. After washing with PBT (PBS containing 0.3% Triton X-100), blocking was
performed in PBTB (PBT with 2% NGA, normal goat serum) for 2 hours at 4°C.
Antibody staining was performed by using a primary rabbit anti-VG at 1/500
(Williams et al., 1991),
primary anti-β-Gal at 1/500, primary anti-ANTP (MAB 8c11) at 1/1000 or
primary anti-EYA at 1/200 (Bonini et al.,
1993) overnight at 4°C. For immunofluorescence detection, a
dichlorotriazinyl amino fluorescein (DTAF)-conjugated donkey anti-IgG (Jackson
ImmunoResearch) antibody was used. The preparations were mounted in
Vectashield (Vector Laboratories) and examined by confocal microscopy using a
Leica (TCS NT) microscope.
Yeast two-hybrid system
A Drosophila third larval instar cDNA library in the pACT vector
(a generous gift from Dr Elledge, Dana-Faber Cancer Institute, Boston, MA) was
screened with an Antp bait that corresponds to the region of aa
280-304 of the ANTP protein, including the YPWM motif and the N-terminal arm
of the HD. The screen was performed as described previously
(Bartel and Fields, 1995) in
L40 yeast cells (Mat, trp1, leu2, his3 LYS2::lexA-lacZ). Around
2x106 clones were screened for β-galactosidase activity.
Quantification of the protein-protein interaction was performed as described
by Bartel and Fields (Bartel and Fields,
1995) after co-transformation in L40 cells. Oligonucleotides
coding for the following peptides were cloned with EcoRI and
BamHI into the pBTM116 vector
(Bartel and Fields, 1995):
LexA-YPWM-N-term, PSPLYPWMRSQFGKCQERKRGRQT;
LexA-AAAA-N-term, PSPLAAAARSQFGKCQERKRGRQT;
LexA-YPWM-, PSPLYPWMRSQFGKCQE; and
LexA-AAAA-, PSPLAAAARSQFGKCQE.
LexA-YPWM-HD was cloned with SmaI and SalI into pBTM116. Amino acids 279-348 of the ANTP protein were used. Bip2-235 was cloned as an XhoI fragment from aa 853-1088 into pACT (from screen).
In all constructs, the splice variant generating the longer linker arm (eight amino acids) was used.
Pull-down experiments and co-immunoprecipitation
A fusion construct ANTP-YPWM-HD-GST was amplified by PCR. This fragment was
subcloned into pGEX-KG (Pharmacia). The resulting GST fusion was expressed in
E. coli and extracted according to Pharmacia's recommendations. For
analyzing protein-protein interactions, 10 µg of GST fusion protein were
incubated with 50 µg of a 50% slurry of glutathione Sepharose 4B beads in
incubation buffer [12 mM HEPES (pH 7.9), 4 mM Tris-HCl (pH 7.9), 50 mM NaCl,
10 mM KCl, 1 mM EDTA, 1 mM DTT, and 1 mM phenylmethylsulfonylfluoride] for 20
minutes at room temperature, washed, and resuspended in a total volume of 30
µl containing 10 µl of [35S]-methionine-labeled rabbit
reticulocyte lysates in incubation buffer for 40 minutes on ice. Beads were
then washed four times with 1 ml of washing buffer [0.5% NP 40, 1 mM EDTA, 20
mM Tris-HCl (pH 8.0), 0.1 M NaCl] at room temperature. Beads were then
recovered in SDS-PAGE loading buffer, and proteins were analyzed by SDS-PAGE
followed by fluorography.
Reticulocyte lysate proteins were produced using the TNT reticulocyte lysate synthesis kit (Promega). Co-immunoprecipitation was performed using third instar larval nuclear extracts. The hemagglutinin (HA)-tagged BIP2 protein was bound to the Anti-HA (3F10) Affinity Matrix (Roche Applied Science). After preabsorbing the Anti-HA Affinity Matrix, 200 µg of nuclear extract was added to the matrix and incubated for 2 hours at 4°C. The matrix was washed twice with buffer I [50 mM Tris HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1 mM PMSF, and protease inhibitors mix]. The precipitate was eluted with SDS sample buffer and analyzed by western blotting, using a monoclonal mouse anti-ANTP antibody (4C3).
Cloning procedure and plasmids
Standard molecular biology methods
(Sambrook et al., 1989) and
yeast protocols (Bartel and Fields,
1995) were used as previously described. The bip2 cDNA
was subcloned into the pBSKII vector with NotI and sequenced using
the Big Dye Terminator Cycle Sequencing Kit and an ABI 373A automated
sequencer (Perkin Elmer/Applied Biosystems). To generate UAS-bip2,
the full-length bip2 cDNA in pBS-SKII was subcloned into the
NotI site of pUAST (Brand and
Perrimon, 1993). To generate UAS-bip2-HA, the C-terminal
200 bp of the bip2 cDNA were replaced by the same bip2 that
was sequenced, fused to the HA coding sequence by PCR. The bip2-HA
cDNA was then subcloned via NotI and Asp718 into pUAST. To
generate pTV2-bip2-SceI-6kb, 6.2 kb of the genomic bip2 region, with
an additional SceI restriction site, was amplified by PCR and
subcloned into the NotI site of the pTV2 vector
(Rong and Golic, 2000).
Generation of bip2 mutants by homologous recombination
bip2 has been mapped to position 102B on the fourth chromosome. We
took w1118; CyO/Sp; hs-Flp, hs-Cre/TM6B,Hu flies and
crossed them to the y1, w1118;
pTV2-bip2-SceI-6kb/pTV2-bip2-SceI-6kb
stock. The offspring were heat shocked (HS) for 1 hour at 37°C at 3 hours
of development. From the heat-shocked offspring, CyO non-TM6b, Hu females that
had lost the w+ marker in the eye were selected. The
females were crossed back to y1, w1118 males.
The progeny was screened for CyO and w+, indicating a
transposition of the mini-white gene from the second chromosome to another
location within the genome. The two flies recovered from 60,000 flies screened
were balanced over ciD, spapol on the fourth
chromosome. The second line with homologous recombination on the fourth
chromosome was recombined with an HS-Flp transgene on the first chromosome,
and crossed to the line Rb_e00710 with a razor Bac vector insertion 1.1 kb
5' to the initiation ATG of the bip2 gene (kindly provided by
Exelixis). Both lines (Rb_e00710 and the line from the homologous
recombination) harbor an FRT sequence oriented in the same direction. The
offspring were heat shocked after 3 hours of development for 1 hour at
37°C. The heat-shocked flies (F1) were crossed back to a fourth chromosome
balancer yw; ciD,
spapol/eyD. F2 flies with dark red eyes
and ciD, spapol were selected and balanced over
ciD, spapol. The recombined chromosomes harbor
the mini-white from of the pTV2 vector, whereas the mini-white gene from the
piggy bac vector was deleted. The two mini-white mutants can be distinguished
by their eye colour (pTV2, dark red; piggy bac, orange), therefore the flies
with the dark eye colour were selected and later screened by PCR for
recombination of the markers.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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)
(Fig. 1A-C). The transformation
of the antenna into leg is not complete, as can be seen in
Fig. 1C. This is owing to the
fact that the driver used (ey-Gal4) is not expressed in the entire
antennal disc. Misexpression of Antp alone by ey-Gal4
promotes antenna-to-leg transformation but does not induce wing development.
Instead it interferes with endogenous eye morphogenesis by inducing apoptosis
(Kurata et al., 2000;
Plaza et al., 2001). By
contrast, activated N signalling alone leads to hyperplasia of the
eye, but no ectopic wings or legs are observed, suggesting that N
activation prevents apoptosis during normal eye development
(Kurata et al., 2000). Kurata
et al. proposed that N signalling modulates the ability of the
precursor cells to respond to different developmental signals, allowing
Antp to promote wing and leg development.
In order to determine whether the YPWM motif of Antp is essential
for the homeotic transformations induced by Antp, we generated
transgenic lines carrying a constitutively active form of the Notch receptor
(Nact) (Fortini et
al., 1993) and a mutated version of the ANTP protein in which the
YPWM motif was substituted by four alanines (ANTPAAAA). None of the
flies expressing UAS-Nact;
UAS-AntpAAAA under the control of the ey-Gal4
enhancer showed any eye-to-wing transformation, whereas about 20% of the flies
showed antenna-to-leg transformation (Fig.
1D,E, Table 1).
These data are in line with the finding that, in the fushi tarazu
gene of the beetle Tribolium (which has a YPWM motif), the AAAA mutation does
not abolish the ability to induce antenna-to-leg transformations in
Drosophila (Lohr and Pick,
2005). Interestingly, legs induced by AntpAAAA
showed second leg identity (Fig.
1E), indicating a strict requirement of the YPWM motif for
eye-to-wing but not antenna- to-second leg transformation. Experiments using a
Distal-less-Gal4 driver indicate the AAAA mutation is capable of
inducing some antenna-to-leg transformation, but to a much lesser extent than
the YPWM construct (R. Fünfschilling, M. Seimiya and W.J.G.,
unpublished).
|
). Kurata et al. previously showed that Antp driven
in combination with Nact in the eye-antennal disc is able
to induce ectopic VG expression. We therefore investigated whether wing
development is induced but not maintained in ey-Gal4;
UAS-Nact; UAS-AntpAAAA larvae.
Immunofluorecence analysis of third instar imaginal discs revealed that
animals carrying both transgenes under the control of ey-Gal4 did not
induce VG (Fig. 1Q,R), in
contrast to the control animals expressing wild-type Antp in
combination with Nact
(Fig. 1O,P). These data
indicate that the YPWM motif is required for the ectopic induction of the wing
selector gene vg in the eye imaginal disc.
The eye-to-wing transformation is specific for the Antp HOX gene
It was shown that homeotic genes in Drosophila have similar
effects when ectopically expressed in the eye-antennal disc
(Casares and Mann, 1998;
Yao et al., 1999). Most HOX
genes are able to transform the distal part of the antenna into leg structures
(Casares et al., 1996;
Kuhn et al., 1993;
Kuziora, 1993;
Mann and Hogness, 1990) by
repressing homothorax (hth)
(Yao et al., 1999). In order
to test the specificity of Antp toward wing and leg development, we
performed gain-of-function experiments with several other homeotic genes, such
as Sex combs reduced (Scr), Ultrabithorax
(Ubx) and Abdominal-B (Abd-B). Misexpression of
UAS-Nact; UAS-Ubx by ey-Gal4 did not
induce any ectopic wing outgrowth in the eye, but it showed antenna-to-leg
transformation, which, however, could not be attributed to a particular
thoracic segment (Fig. 1H-J).
The same results were obtained with Scr and Abd-B (data not
shown). In addition, we found that changing the DNA-binding specificity of the
Antp HD (AntpQ50K) abolishes the capacity of
Antp to induce ectopic wing tissue in the eye and also reduces the
capacity to transform the antenna into a leg. Only an arista-to-tarsus
transformation is observed (Fig.
1F,G). When an Antp gene lacking the HD is co-expressed
with Nact, these flies show the same phenotype as
ey-Gal4; UAS-Nact flies, i.e. enlarged eyes (data
not shown). Thus, among the different HOX genes tested, only Antp is
able to induce ectopic wing development, a transformation dependent on the
YPWM motif and the DNA-binding specificity of the protein.
Wing induction is inhibited by the overexpression of exd
So far, EXD is the only HOX co-factor known to bind the YPWM motif. To test
whether exd is required to promote wing versus leg development, we
co-expressed exd in combination with the wild-type Antp or
the AntpAAAA transgene in the presence of
Nact, as described above. We found that only 2% of flies
carrying the ey-Gal4; UAS-Nact;
UAS-Antp; UAS-exd transgenes showed wing structures in the
eye region, as compared with 40% of flies without exd
(Table 1). Similarly,
exd inhibited the antenna-to-leg transformation, as only 3% of the
flies expressing exd showed antenna-to-leg transformation compared
with 28% without exd (Table
1). Furthermore, exd also inhibits the antenna-to-leg
transformation induced by AntpAAAA from 19% to 4.5%
(Table 1). This inhibition is
caused by EXD, which is localized in the nuclei as shown by antibody staining.
Both proteins, EXD and ANTP, are found to be co-expressed in the nucleus of
the eye-antennal disc cells (see Fig. S1 in the supplementary material). The
suppression of the antenna-to-leg transformation by exd is in line
with previous studies that show that nuclear EXD is incompatible with distal
leg development (Casares and Mann,
1998). We further do not attribute the observed effect to a
dilution of the GAL4 protein by the addition of a third UAS transgene into the
system, as the addition of an UAS-bip2 transgene instead of
UAS-exd enhances the eye-to-wing transformation (see below). As the
co-expression of exd in combination with Antp and
Nact reduces the frequency of eye-to-wing transformations,
these results indicate that exd is not acting in combination with
Antp to induce ectopic wing development.
Identification of a new ANTP-interacting protein
Because the YPWM motif of ANTP plays an essential role in ectopic wing
development, we screened for proteins that specifically interact with the ANTP
YPWM motif using the yeast two-hybrid system
(Bartel and Fields, 1995). We
screened a third instar Drosophila cDNA library fused to the Gal4
activation domain with a bait composed of the LexA DNA-binding domain fused to
the ANTP YPWM motif and the N-terminal arm of the HD (LexA-YPWM-N-term)
(Fig. 2A). We identified two
identical cDNA clones (BIP2-235; amino acid 853-1088) of the gene
bip2 (Gangloff et al.,
2001). The clone fused to the Gal4 activation domain interacted
specifically with the ANTP bait and not with the empty vector
(Fig. 2B).
BIP2 specifically interacts with the YPWM motif
To identify the protein domain of ANTP that is essential for the
interaction with BIP2, several deletion constructs of ANTP were assayed for
interaction (Fig. 2A). The
interaction between the YPWM full-length ANTP-HD (LexA-YPWM-HD, aa 279-348)
and BIP2-235 is similar to the interaction observed using the ANTP YPWM-N bait
used in the screen (Fig. 2B,C,
compare lanes 1 and 2). Then, we compared the relative interactions of various
ANTP baits carrying deletions and/or substitutions of the N-terminal arm of
the HD and/or the YPWM motif to BIP2-235. Substituting the YPWM motif of the
LexA-YPWM-N-term bait by four alanines (LexA-AAAA-N-term) reduced the
interaction (Fig. 2B,C, compare
lanes 2 and 3), whereas deleting the N-terminal arm (LexA-YPWM-) had only a
mild effect on the interaction (Fig.
2B,C, compare lanes 2 and 4). By contrast, substituting the YPWM
motif and deleting the N-terminal arm abolished the interaction
(Fig. 2C, lane 5). Although the
yeast two-hybrid analysis shows that the major interaction occurs with the
YPWM motif and that the N-terminal arm of the HD exerts a minor effect only,
BIP2-235 is able to interact with the complete ANTP HD lacking the YPWM motif,
indicating additional interaction surfaces (data not shown).
BIP2 forms a complex with ANTP depending on the YPWM motif in vivo
In order to confirm the interaction found in vitro between ANTP and BIP2,
and its YPWM-motif dependence, we used a co-immunoprecipitation assay. As
shown in Fig. 2E, we were able
to co-immunoprecipitate ANTP protein (detected with a anti-ANTP antibody) with
BIP2 by using an anti-HA-affinity-matrix aimed at isolating a hemagglutinin
(HA)-tagged BIP2 protein (BIP2-HA) from larval nuclear extracts
(overexpressing wild-type Antp and bip2-HA upon heat shock
treatment; HS-Gal4>UAS-Antp; UAS-bip2-HA;
Fig. 2E, lane YHA). When the
YPWM motif mutated version of ANTP (ANTPAAAA) is co-expressed, no
complex between this modified ANTP protein and BIP2 is formed
(Fig. 2E, lane AHA), which
shows an in vivo requirement of the YPWM motif for the ANTP-BIP2 complex
formation. As expected the ANTPAAAA protein can be detected in the
supernatant lane (Fig. 2E, lane
AHA of the supernatant). To demonstrate the specificity of the interaction in
the cell extracts, we co-expressed the wild-type and mutated ANTP protein in
combination with BIP2 not fused to HA (HS-Gal4>UAS-Antp;
UAS-Bip2 and HS-Gal4>UAS-AntpAAAA;
UAS-Bip2). As seen in Fig.
2E lanes Y and A, the weak protein detection represents
non-specific binding to the anti-HA-affinity matrix.
|
bip2 acts as a co-factor of Antp by enhancing eye-to-wing transformation
BIP2 was first identified as a Bric-à-brac (BAB)-interacting protein
encoding a TATA-box-binding protein associated factor (TAF)
(Albright and Tjian, 2000),
also referred as dTAFII3 or dTAFII155
(Gangloff et al., 2001). BIP2
is a member of the TFIID complex for transcription initiation and is the
Drosophila homolog of TAF3. The BIP2 transcript and protein are
ubiquitously expressed during embryogenesis and are widely expressed in all
third instar imaginal discs (Gangloff et
al., 2001).
|
)
indicate a broad expression of BIP2 throughout development, including a
maternal contribution, suggesting several functions played by this gene in
various cells at different stages, rendering the analysis difficult. As no
bip2 mutants were available, we decided to generate mutants by the
homologous recombination technique (Rong
and Golic, 2000). The resulting bip2 mutants have a 1.2
kb (-1.1 kb 5' to +152 bp 3' of the start ATG) deletion around the
start ATG, deleting the first exon harbouring the Histone Fold Domain (HFD)
(data not shown). The two alleles, bip23 and
bip24, obtained are late embryonic or first instar larval
recessive lethals without any obvious morphological defects (data not shown).
The analysis of cuticles did not reveal any visible phenotype, as observed in
HOX or exd mutations, probably because of the strong maternal
contribution (data not shown). As bip2 lies at position 102B on the
fourth chromosome, we were not able to perform a germline or somatic clonal
analysis, instead we used a gain-of-function approach to address the question
of a functional relevance of bip2 in Antp-induced
transformations. Upon ectopic co-expression of bip2 in combination
with Antp and Nact, we found that co-expression
of bip2 strongly enhanced the penetrance of eye-to-wing
transformation, whereas exd had the opposite effect
(Table 1). About 80% of the
flies carrying ey-Gal4; UAS-Nact;
UAS-Antp; UAS-bip2 transgenes showed eye-to-wing
transformation compared with 40% without co-expression of bip2
(Table 1). The opposite effect
was observed when comparing the antenna-to-leg transformation. The
co-expression of bip2 in combination with Antp and
Nact reduced the fraction of flies showing antenna-to-leg
transformation from 28% to 13% (Table
1). No obvious effect of bip2 could be observed in
combination with the ANTPAAAA protein, neither on eye-to-wing nor
on antenna-to-leg transformation (Table
1), indicating a requirement of the YPWM motif for the ANTP/BIP2
interaction and for the specificity observed by adding BIP2 protein. Owing to
the fact that opposite effects are seen in combination with wild-type ANTP,
and no effect with ANTPAAAA, rules out an unspecific effect of BIP2
by either influencing the transcriptional machinery or sequestering the Gal4
protein. We conclude from these results that the phenotypes observed are due
to the interaction of Antp with bip2, as transgenic flies
expressing UAS-Nact; UAS-bip2 under the control
of ey-Gal4 showed the same phenotype as flies expressing
UAS-Nact alone (data not shown). Interestingly, the
ectopic wing formation is not entirely dependent on an activated form of the
N receptor. Using the eye-specific OK-107-Gal4 driver to express
Antp ectopically in the eye disc, we found 8% (10/132) of flies
having an eye-to-wing transformation (Fig.
3G), in addition to the previously described eye-reduction
phenotype (Fig. 3E,F). The
penetrance of the eye-to-wing transformation is increased upon ectopic
co-expression of bip2 to 24% (27/111). No ectopic wings could be
observed when the YPWM motif-mutated (AAAA) ANTP protein was expressed by the
OK-107 enhancer, instead the flies showed a strong eye-reduction phenotype
with a similar penetrance to the wild-type Antp transgene (data not
shown). We can exclude the possibility that the YPWM motif dependence to
induce eye-to-wing transformation is due to low protein levels, because the
line shows a similar penetrance of the eye-reduction phenotypes as the
wild-type Antp transgenic lines. Although we have never seen any
ectopic wing formation using an ANTPAAAA mutant protein, we are not
able to entirely exclude a quantitative rather than a qualitative effect of
the YPWM motif. In order to address this question, we repeated the above
described experiment using an additional UAS-Gal4 insert to enhance
the ectopic protein expression (OK-107-Gal4; UAS-Gal4;
UAS-AntpAAAA), but could never observe any eye-to-wing
transformation. Instead a high degree of lethality was observed making the
analysis difficult. These data strongly suggest that bip2 acts as a
co-factor of Antp promoting ectopic wing development.
The Antp allele AntpCephalothorax allele shows eye-to-wing transformation
To find out whether the ANTP/BIP2 complex plays a role in vivo, we searched
through the literature for Antp alleles inducing ectopic wing
transformations. Scott et al. found some transformations caused by the
AntpCephalothorax (AntpCtx) allele
corresponding to the phenotypes obtained by co-expression of Antp in
combination with Nact
(Fig. 3A-D)
(Scott et al., 1983), namely
ectopic wings and ectopic thoracic outgrowths on the dorsal rim of the eye
(Fig. 3A-D), head
capsule-to-thorax (Fig. 5B) and
antenna-to-leg transformations, and eye-reduction
(Fig. 5B,
Fig. 3B, data not shown).
AntpCtx is an Antp gain-of-function allele with a
chromosomal translocation between the Antp locus 84B1,2 and 35B,
where Su(H) maps to (Scott et al.,
1983). In order to characterize the molecular events involved in
these transformations, we tested whether the Notch signalling pathway
is activated in these transformed discs. Using a Su(H)-reporter construct
[Su(H)-lacZ] indicating the activation of the Notch pathway
(Furriols and Bray, 2001), we
found the N signalling pathway ectopically activated in transformed
AntpCtx eye imaginal discs
(Fig. 4A,B). Additionally,
ectopic ANTP and VG protein can be observed in the dorsal part of
AntpCtx eye imaginal discs
(Fig. 4C), where the ectopic
wings will form (Fig. 3B,
Fig. 5A). Consistent with the
adult phenotype, the neuronal identity marked by the Eyes absent (EYA) protein
is repressed in the transformed part of the discs, marked by the presence of
VG protein (Fig. 4D,E).
Interestingly, the repression of neural fate is non-cell autonomous to
vg expressing cells, indicating that cells adjacent to the VG
expressing clones are also transformed.
|
;
Couso et al., 1994;
Jang et al., 2003;
Phillips and Whittle, 1993).
As seen in Fig. 4F, reduced eye
discs show broad ectopic eyg expression that is not co-expressed with
ectopic VG, marking thoracic/notum identity. The co-expression of VG and
wg-lacZ seen in Fig.
4H illustrates a transformation of the eye disc toward wing pouch
identity. We find co-expression of SRF-β-Gal, a wing pouch marker, in the
ectopic VG domain (data not shown). In agreement with the morphology of the
eye imaginal disc, head capsule toward thorax transformation, marked by strong
eyg expression, is strongly linked to eye-loss phenotype, as
indicated by a reduced disc size (Fig.
5B). By contrast, ectopic wings, marked by VG and WG
co-expression, are associated with slightly reduced eyes only and eye imaginal
discs (Fig. 3B,D,G,
Fig. 5A).
We further analyzed the expression of another signaling pathway, the
decapentaplegic (dpp) pathway. Transdetermination
experiments have revealed `weak points' representing cells that are plastic
and capable of altering their normal selector gene expression
(Maves and Schubiger, 2003).
These weak points can be defined by strong dpp expression. When
wg is ubiquitously expressed in leg discs, cells with strong
dpp expression (dorsal cells) are able to change fate and express VG
protein, representing a leg-to-wing transdetermination
(Maves and Schubiger, 2003).
By analyzing the AntpCtx eye-antennal imaginal disc for
ectopic wg and dpp expression, we could observe domains
co-expressing both signalling molecules
(Fig. 4J,K), indicating a
general eye-to-wing transformation (transdetermination) induced by
Antp.
These data suggest that the eye imaginal disc in AntpCtx flies is transformed into different parts of the wing disc, indicating a more general homeotic transformation toward T2.
bip2 genetically interacts with AntpCtx
As the AntpCtx allele shows ectopic wing tissue on the
head, we tested whether the newly created loss-of-function bip2
mutant genetically interacts with the AntpCtx allele, by
reducing the frequency of eye-to-wing transformations. We compared
AntpCtx/+;ciD/+ to
AntpCtx/+;bip24/+ flies. After
crossing AntpCtx/CyO females to yw;
bip24/ciD males, 14% of the
AntpCtx/+; ciD/+ flies show ectopic wings on
the head as compared with 4% in AntpCtx/+;
bip24/+ transheterozygous flies
(Table 2,
Fig. 5C). bip2 also
influences the eye-reduction phenotype. Comparing the different
transheterozygous flies, we could not only observe a change in the penetrance
of ectopic wing formation, but also a change in head phenotype (represented in
Fig. 5A-C). Flies showing an
ectopic wing on the head almost always show a normal or slightly reduced eye,
whereas flies with a head capsule-to-thorax transformation mostly have reduced
or lost eyes (Fig. 5A,B). In
order to quantify these phenotypes, we monitored the eye phenotypes of the
above mentioned transheterozygous flies. Reducing the gene dosage of
bip2 increases the eye reduction phenotype.
AntpCtx/+; bip24/+ flies show stronger
eye-reduction/eye-loss phenotypes than do AntpCtx/+;
ciD/+ flies. Ninety percent of the eyes of
AntpCtx/+; ciD/+ flies show a normal
to weakly reduced eye phenotype, whereas only 34% of the eyes of
AntpCtx/+; bip24/+ flies show a normal
eye phenotype. By contrast, 34% of the eyes of AntpCtx/+;
bip24/+ flies show an eye-loss phenotype, compared with 2%
without the bip24 mutation
(Table 2,
Fig. 5C). Similar results were
obtained in the reciprocal cross: yw; bip24/ciD
females x AntpCtx/CyO males (data not shown). In
conclusion, the bip2 loss-of-function mutation reduces the frequency
of AntpCtx flies showing ectopic wing outgrowth on the
head and enhances the Antp-induced eye reduction phenotype - which we
used as a measure for strong head capsule-to-thorax transformation. Consistent
with the reduction of ectopic wings in a bip24 mutant
background, overexpression experiments of bip2 show an increase of
ectopic wing outgrowth in combination with Antp and
Nact (see above).
|
|
). We
could not observe any effect of bip24 on the
Antp73b allele, in agreement with the gain-of-function
experiment with bip2 counteracting the antenna-to-leg transformation
by Antp. |
DISCUSSION |
|---|
|
TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
) and for
the QA motif of UBX (Hittinger et al.,
2005).
The addition of the well-known HOX co-factor exd, that has been
shown to bind via the YPWM motif, antagonizes the eye-to-wing transformation,
indicating a YPWM-motif-dependent Antp function, independent of
exd. We cannot exclude the possibility that exd has an
Antp-independent effect by repressing wing development, as is the
case for leg development (Casares and
Mann, 1998), but the overexpression of exd fused to a
nuclear localization signal does not interfere with endogenous wing
development (Jaw et al.,
2000). We cannot distinguish whether deleting the YPWM motif of
Antp changes its DNA-binding selectivity or whether Antp
loses its transactivation potential, as the direct targets of Antp
genes in the eye-to-wing transformation remain to be identified. Nevertheless,
we favour the later possibility, although it was shown that mutating the YPWM
motif of HOXA5 does not interfere with the transcriptional activity of the
protein (Zhao et al.,
1996).
We found bip2 acting as an Antp co-factor for ectopic
wing formation, linking Antp to an activating TFIID complex and to
the basal transcriptional machinery. Previously it has been shown that HOX
gene activity regulation might play in important role in HOX-dependent gene
regulation (Li and McGinnis,
1999; Li et al.,
1999; Merabet et al.,
2003). bip2 might also provide target gene specificity by
linking Antp to a specific TFIID complex, which might confer
specificity through promoter selectivity, as was shown for other TAF-complexes
(Verrijzer and Tjian,
1996).
In summary, our data indicate that the YPWM motif is a more generally used protein-protein interaction interface interacting with at least two, but probably more protein co-factors, judging from the numerous exd-independent HOX functions that have been found.
bip2 acts as a co-factor of Antp promoting eye-to-wing transformation
The YPWM-motif dependence of the Antp-specific eye-to-wing
transformation implies the existence of a novel YPWM-motif-interacting
protein, as the YPWM motif is considered to be a protein-interaction domain.
Using the yeast two-hybrid system, we found a new ANTP-interacting protein,
encoded by the bip2 gene
(Gangloff et al., 2001),
Drosophila TBP-associated factor 3 (dTafII3/dTAFII155;
BIP2 - FlyBase). Several lines of evidence indicate that bip2 might
be a novel Antp co-factor interacting with the YPWM motif. (1) In our
gain-of-function experiments, bip2 behaves as an Antp
co-factor promoting ectopic wing development, and the bip2
loss-of-function mutation genetically interacts with the Antp allele
AntpCtx, reducing the frequency of eye-to-wing
transformations. (2) bip2 acts as a co-factor for an Antp
function requiring the YPWM motif. (3) BIP2 interacts in vitro with the YPWM
motif in a yeast two-hybrid assay and shows an in vivo requirement of the YPWM
motif for the ANTP-BIP2 interaction in a co-immunoprecipitation assay.
bip2 (dTAFII3) is a member of the TBP-associated TFIID
complex in the basal transcriptional machinery, and belongs to the class of
histone-like TATA-binding protein (TBP)-associated factors (TAF) with two
homologues in yeast, humans and mice
(Gangloff et al., 2001). The
bip2 gene codes for a protein with two distinct domains, a Histone
Fold Domain (HFD) at the N terminus and a Plant Homeodomain (PHD) finger at
the C terminus. The HFD is a domain initially found in histones involved in
the formation of histone dimers (Aasland et
al., 1995), whereas the PHD has been recently shown to
specifically interact with three-methylated histone H3 at lysine 4
(Li et al., 2006;
Pena et al., 2006;
Shi et al., 2006;
Wysocka et al., 2006). BIP2
forms a histone-like dimer with TAF10 (dTAFII24)
(Gangloff et al., 2001). This
dimer formation is conserved from yeast to humans
(Gangloff et al., 2001).
bip2 and its homologues have been identified as members of the
TBP-containing TFIID complex (Gangloff et
al., 2001), linking ANTP to the basal transcriptional machinery.
But, BIP2 might also be a part of a TBP-free TAF-containing complex (TFTC), a
histone acetyl transferase complex (HAT). The human homologue of BIP2 and
TAF10, and TAF10 itself are found to co-immunoprecipitate with GCN5 (PCAF -
FlyBase), the acetyl transferase of the TFTC HAT complex
(Georgieva et al., 2000;
Grant et al., 1998;
Martinez et al., 1998;
Ogryzko et al., 1998;
Wieczorek et al., 1998), and
BIP2 harbours a PHD domain implicated in reading specific histone codes
(Li et al., 2006;
Pena et al., 2006;
Shi et al., 2006;
Wysocka et al., 2006).
Furthermore, Drosophila has two paralogous genes encoding TAF10
homologues, Taf10 and Taf10b, which are differentially
expressed during development (Georgieva et
al., 2000). BIP2 specifically forms a dimer with TAF10 and not
with TAF10b (Gangloff et al.,
2001); TAF10 was found to be present in both TFIID and TFTC-like
complexes, whereas TAF10b was only identified in TFIID complexes
(Georgieva et al., 2000).
These results raise the possibility of ANTP being linked to a histone
acetylase complex. The link unravelled between Antp and bip2
raises numerous questions, including which complex incorporating Antp
is present to perform its wing promoting function? Interestingly, Katsuyama
and co-workers found a novel gene winged eye (wge)
implicated in the eye-to-wing transformation
(Katsuyama et al., 2005).
wge seems to be downstream of Antp in the developmental
process of eye-to-wing transformation
(Katsuyama et al., 2005).
wge codes for a bromo-adjacent homology domain (BAH)-containing
protein (Katsuyama et al.,
2005). The BAH domain has frequently been associated with other
domains, such as bromodomains, PHD fingers, and Suppressor of variegation 3-9,
Enhancer of zeste and Trithorax (SET) domains, in proteins that are suggested
to be involved in the epigenetic regulation of gene expression
(Callebaut et al., 1999). This
indicates that epigenetic regulation of so far unknown genes is involved in
eye-to-wing transformation.
Antp is able to induce ectopic wings
It has previously been shown that Antp in combination with
Nact is able to induce ectopic wings by transforming
eye-to-wing tissue (Kurata et al.,
2000). Although endogenous wing development is considered to be
independent of Antp (Carroll et
al., 1995), we found that Antp is the only HOX gene
tested so far that is able to transform the eye into wing, which is in line
with the fact that Antp specifies the entire second thoracic segment.
Furthermore, AntpCtx is the only homeotic
gain-of-function allele found that induces ectopic wings on the head
(Scott et al., 1983).
Several lines of evidence indicate that N supports Antp
in inducing ectopic wings, by preventing eye cells from undergoing apoptosis
and in allowing them to adopt a new developmental fate. First, wings formed by
ectopic expression of Antp in combination with
Nact, or wings found on AntpCtx heads,
show the same characteristic triple row of bristles at the wing margin
(Fig. 4B,C). These bristles are
found only when vg is ectopically co-expressed in combination with
wingless (wg), not when in combination with
Nact (Baena-Lopez and
Garcia-Bellido, 2003). Second, we found that N alone does
not induce ectopic wings, and that eye-to-wing transformation can also be
achieved without the action of N, by using another eye-specific
driver, OK-107-Gal4 (see Hauck et al.,
1999), indicating that N is not absolutely required for
ectopic wing induction. Using different markers for different parts of the
wing disc, we found parts of the eye disc to be transformed into most wing
disc identities from wing pouch to notum, indicating an eye-to-dorsal T2
transformation, rather than the eye-to-wing pouch transformation seen in adult
flies.
The known HOX co-factors exd and hth code for DNA-binding proteins that have been shown to increase DNA-binding specificity. bip2, however, encodes a member of the basal transcriptional machinery without any DNA-binding capacity, indicating a different mechanism of action, i.e. by linking Antp directly to the transcriptional machinery. In summary, we propose that ANTP interacts directly with BIP2, activating, in turn, a subset of genes that are implicated in wing development.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/9/1669/DC1
|
ACKNOWLEDGMENTS |
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