1 Institut de Génétique Humaine (UPR 1142). 141 rue de la
Cardonille, 34396 Montpellier, France
2 Laboratoire de Génétique et Physiologie du Développement
(UMR 6545), IBDM, Parc Scientifique de Luminy, 13288 Marseille, Cedex 9,
France
Author for correspondence (e-mail:
fmaschat{at}igh.cnrs.fr)
Accepted 18 December 2002
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SUMMARY |
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Key words: Engrailed, Chromatin immunoprecipitation, In vivo targets, Drosophila
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INTRODUCTION |
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To address this issue, we decided to identify genomic fragments that are
bound in vivo by the homeoprotein Engrailed in Drosophila embryos.
engrailed is involved in posterior cell identity, as well as in the
maintenance of the anteroposterior (AP) boundary throughout development
(Lawrence and Morata, 1976;
Kornberg, 1981
;
Maschat et al., 1998
).
Furthermore, engrailed is expressed in a subset of neuroblasts and
neurons in the developing central nervous system and in the brain. This
suggests a role for engrailed during neurogenesis that is believed to
be highly conserved during evolution (Bhat
and Schedl, 1997
; Hanks et
al., 1998
; Siegler and Jia,
1999
; Simon et al.,
2001
).
We have previously used UV crosslinking and in vivo chromatin
immunoprecipitation to analyze Engrailed-binding sequences associated with
particular genomic regions such as polyhomeotic or
ß3-tubulin genes (Serrano et
al., 1995; Serrano et al.,
1997
). We present a similar approach, performed on a larger scale,
leading to a genome-wide view of direct Engrailed-binding loci in embryos. UV
light or formaldehyde crosslinking are currently used to purify protein-DNA
complexes, and to isolate specific binding fragments
(Graba et al., 1992
;
Serrano et al., 1995
;
Serrano et al., 1997
;
Liang and Biggin, 1998
;
Cavalli et al., 1999
;
Toth and Biggin, 2000
;
Weinmann et al., 2001
;
Weinmann et al., 2002
).
However, UV light is believed to be more efficient in fixing proteins that are
directly bound to DNA (Toth and Biggin,
2000
).
In the present report, we constructed a library enriched in genomic sequences that bind Engrailed protein in Drosophila embryos, by using UV crosslinking and chromatin immunoprecipitation (UV-X-ChIP). Systematic sequencing of the recovered clones led to the identification of 203 potential direct targets of engrailed and evidence is presented to show that some of them represent bona fide engrailed targets.
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MATERIALS AND METHODS |
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The 1A4-GFP strain was made for the purpose of this study, by inserting 1A4
DNA fragment (either as a monomer or a trimer) into the WH.GFP (obtained from
B. Bello, MRC, Mill Hill, UK). This vector corresponds to a mini white-based
P-element vector with multiple cloning sites upstream of the hsp70
minimal promoter from RHT vector (Bello et
al., 1998) and the GFP F64L/65T-drosomycin poly A signal
(Levashina et al., 1998
).
Different transgenic lines were obtained with both 1 A4 monomer and trimer and
showed the same GFP expression pattern as described in
Fig. 5.
|
Proteins
A Schneider two-cell line, transformed with a gene fusion where the
engrailed cDNA was placed under the control of the hsp70
promoter, was grown at 25°C (Gay et
al., 1988). To induce engrailed expression, cells were
placed at 37°C for 45 minutes, followed by 2 hours at 24°C. Soluble
nuclear extract (referred as to HS-EN) was prepared as described
(Gay et al., 1988
). A typical
extract contained 2.5 mg/ml of proteins, of which 2% is Engrailed (estimated
from western analysis in comparison with FPLC Engrailed purified proteins).
HS-EN protein has been used in in vitro immunoprecipitation and in gel shift
assays.
Antibodies
Anti-Frizzled 2 antibody is a mouse monoclonal antibody
(Cadigan et al., 1998). The
following anti-Engrailed antibodies were used: monoclonal antibody 4F11 for
immunoprecipitation and super-shift assays, and rabbit polyclonal antibody
raised against the entire Engrailed protein for immunostaining
(Serrano et al., 1995
).
Anti-HA 12CA5 is a mouse monoclonal (Roche) and is monitored using a
biotinylated secondary antibody, prior to HRP detection (Vector, Vectastain).
Cy3-conjugated anti rabbit is from Immunotech.
cDNA
cDNAs used in this study were obtained from different laboratories:
frizzled 2 (Cadigan et al.,
1998); branchless
(Ribeiro et al., 2002
);
frazzled (Kolodziej et al.,
1996
) and hibris
(Artero et al., 2001
).
Construction of the library
A detailed protocol of X-ChIP is available at
http://www.igh.cnrs.fr/equip/WebFM/.
Briefly, nuclei were prepared from a 0-16 hour Oregon R embryo collection.
Purified nuclei were irradiated with UV light Stratalinker (254 nm at 10 cm
for 10 minutes) in order to freeze DNA-protein interactions. Chromatin was
recovered from these nuclei by using detergents. Free proteins were separated
from the rest of the chromatin by CsCl ultra centrifugation. The supernatant
was dialyzed against TE buffer overnight. Chromatin was sonicated in order to
shear DNA to 0.1-3.0 kb average length. We usually obtained 100 µg of
chromatin from 1 g of embryos. Around 40 µg of chromatin were used for each
immunoprecipitation experiment. Chromatin was first incubated for 1 hour at
4°C in the presence of 100 µl of protein A sepharose CL-4B resin
(Amersham Pharmacia). The chromatin was then incubated overnight at 4°C,
with either 50 µl of resin coupled to 4F11 anti-Engrailed antibody (which
corresponds to `EN probe') or 50 µl of resin with no antibody (`background
probe'). After several washes, chromatin bound on the resin was eluted in the
presence of 4% Sarkosyl. Samples were dialyzed, and DNA ends were repaired by
Klenow before further purification. Samples were treated with RNase,
proteinase K and phenol/chloroform extracted before precipitation in the
presence of 20 µg glycogen. Linkers containing cloning sites were added and
DNA was amplified by PCR, using a primer that covers the linker. Typically,
10-20 µg of amplified DNA were recovered. At that stage, DNA can be either
labeled by random priming to be used as a probe on a Southern blot or
processed for further purification. Indeed, to construct the library, we
performed an additional in vitro immunopurification using a quarter of the PCR
amplification after the `in vivo' step, under the conditions described by
Serrano et al. (Serrano et al.,
1995). DNA was incubated in the presence of 50 µl resin where
4F11 anti-Engrailed antibody was bound, as well as Engrailed protein isolated
from 1 mg of HS-EN cell culture nuclear extracts (containing
20 µg of
Engrailed protein). After overnight incubation at 4°C, resin was washed
and DNA was eluted in the presence of 1 M KCl. After phenol/chloroform
extraction and precipitation in the presence of glycogen, DNA was PCR
amplified using a primer that covers the linker. DNA was then
phenol/chloroform extracted and precipitated, digested using HindIII
and cloned into pBluescript KS+. Each clone was
sequenced.
Southern blots
For each of the 315 clones of the library, 1 µg of plasmid DNA was
digested by HindIII, separated on 1% agarose gel in 0.5x TBE
and transferred onto GeneScreen Plus membranes (NEN Life Science). Membranes
were hybridized and washed following manufacturer instructions. Probes were
prepared by 32P labeling of 75 ng PCR amplified DNA (isolated after
`in vivo' immunoprecipitation), using the Rediprime kit labeling system
(Amersham Pharmacia). Signals have been quantified using a phosphoimager.
Gel shift assays
DNA probes were synthesized by PCR amplification, using specific primers
that were 32P end labeled using T4 kinase. After gel purification,
binding assays were performed in the presence of 1-5 ng labeled DNA,
corresponding to 2000 cpm. Different quantities of HS-EN protein, isolated
from cell culture nuclear extracts, were incubated with DNA probe for 30
minutes at 4°C in 10 µl of 25 mM HEPES (pH 7.6), 10% glycerol, 100 mM
KCl, 1 mM DTT, 1% PVA, 1% NP40, 0.1% BSA and 200 ng of poly(dI:dC).
DNA-protein complexes were resolved on 6% native polyacrylamide minigels in
0.5x TBE buffer (pH 8.3). For competition experiments, 750 ng unlabelled
competitor DNA were added to the mix, and incubated with the protein and
labeled DNA for 30 minutes at 4°C, before loading on gel. For supershift
experiments, 4F11 antibody was incubated together with the labeled DNA for 30
minutes at 4°C, in the absence (-) or in the presence (+) of HS-EN
protein, before loading on gel.
RNA in situ hybridization
Embryo fixation and in situ hybridization using DIG labeled antisense RNA
probes were performed as described previously
(Alexandre et al., 1996).
Dissected larvae were fixed for 20 minutes in fixation buffer [30 mM PIPES (pH
7.4), 160 mM KCl, 40 mM NaCl, 4 mM EGTA, 1 mM spermidine, 0.4 mM spermine,
0.2% ß-mercaptoethanol, 0.1% Triton X-100 and 4% paraformaldehyde], then
washed four times in PBS + 0.1% Tween. The conditions for in situ
hybridization were the same as for embryos. For double staining in embryos, in
situ hybridization was performed first with an alkaline phosphatase-based
detection system (Roche), followed by incubation with polyclonal
anti-Engrailed antibody (dilution 1:300) overnight at 4°C. Detection of
the immune signal was carried out with biotinylated secondary antibody, prior
to HRP detection (Vectastain). Embryos were dehydrated and mounted in Canada
Balsam for observation.
Immunostaining and in situ hybridization of polytene chromosomes
Squashes and hybridization were performed in tramtrack mutant
background (ttk804), allowing engrailed expression in the
salivary glands, as described by Serrano et al.
(Serrano et al., 1995), with
the following modifications: the 1A4-GFP DNA, labeled using Bionick labeling
system (Invitrogen), was used as a probe and detected using fluorescein
anti-biotin antibody (Vector) (1:200). Polyclonal anti-Engrailed antibody
(1:200), secondary detected by Cy3 anti-rabbit antibody (1:200) has been used
to identify Engrailed-binding sites. Chromosomal banding was detected with
DAPI.
Computational analysis
A series of Perl programs were specifically written to analyze UV-X-ChIP
datasets (D. Martin, F. M. and B. J., unpublished). The sequences of all
immunoprecipitated fragments were automatically compared with the
Drosophila genome sequence (BDGP), using the blastn program
(Altschul et al., 1990). The
output was automatically treated using scripts in order to remove
contaminating vector or non-Drosophila sequences and to detect
repeated elements or chimeric fragments. Using this approach, 357 distinct
Engrailed-binding loci have been identified, from 542 clones sequenced.
Functional assignments for potential target genes were automatically performed
through a script querying Gene Ontology (GO) terms associated to them in
FlyBase (Ashburner et al.,
2000
).
In order to discover over-represented motifs in these sequences, only
clones without internal HindIII restriction site were considered.
These genomic sequences might contain all the information necessary for
Engrailed recognition. Out of the 203 positive clones, 107 sequences agreed
with this criteria and were analyzed using the MEME algorithm, according to
Bailey and Elkan (Bailey and Elkan,
1995). This led to the identification of 49 related motifs present
in 204 hits and compiled in a position weight matrix presented on
Fig. 2A.
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RESULTS |
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To determine if these fragments were specifically enriched through immunoprecipitation, and do not simply represent nonspecific binding to the protein A sepharose resin, we performed Southern blots on 323 clones. We compared hybridization signal intensity with EN probe and a Background probe (see Materials and Methods) (Fig. 1A). With this assay, only the fragments for which the ratio EN probe/Background probe was higher than 2 were considered for further analysis. This was the case for 65% of the 315 intronic and intergenic fragments, whereas the eight DNA fragments lying in exonic sequences showed signals comparable with both probes (data not shown). As an example, Fig. 1B shows the results for 14 positive clones. Thus, 203 clones isolated in the library correspond to DNA that was specifically enriched by UV-X ChIP.
Localization of the Engrailed-binding fragments within the
Drosophila genome and assignment for potential target genes
In order to localize precisely the 203 Engrailed-binding fragments, the
corresponding sequences were compared with the published Drosophila
genome sequence (Adams et al.,
2000). This allowed the identification of genes in the same
location, which were categorized according to Gene Ontology (GO) annotation
(see Materials and Methods, Table
1 and
http://www.igh.cnrs.fr/equip/WebFM/).
|
Forty-seven percent of the fragments were localized within gene introns. In this case, we assumed that the corresponding intron is a part of the engrailed regulated target gene. Fifty-three percent were present in intergenic regions. In this case, we restricted our analysis to the nearest transcription unit, whatever its orientation and its distance with respect to the Engrailed-binding fragment. Half of the intergenic fragments are localized at less than 5 kb upstream of the genes, suggesting that the Engrailed-binding fragment may be a part of their promoter region. In rare cases (5%), when the Engrailed-binding fragment lies between two transcription units among which only one encodes a known function, we considered the latter as the putative target.
In 55% of the cases, the Engrailed-binding fragments could be associated
with a gene whose function is known or that contains a recognizable protein
domain. In all the other cases (45%), the binding fragments were associated to
genes with an unknown function, which is approximately the ratio of this
category in the Drosophila genome
(Adams et al., 2000). For an
overview, 81 genes of known or predicted function are presented here and have
been grouped into functional classes (Table
1). The other 30 genes encode proteins with recognizable domains,
the function of which is unknown, and have not been listed here.
According to GO annotation, the majority of these genes are involved in
cell communication and developmental processes
(Table 1). As expected from
previous work, potential Engrailed targets identified using this approach
include genes that are involved in the establishment and the maintenance of
the AP axis body (Kornberg,
1981; Vincent and O'Farrell,
1992
; Serrano et al.,
1995
; Alexandre and Vincent,
2002
). We also identified several genes involved in wing
development (Hidalgo, 1994
;
Maschat et al., 1998
),
tracheal development, muscle development
(Serrano et al., 1997
) and
axon guidance (Siegler and Jia,
1999
). Furthermore, different categories of genes encoding
proteins involved in signal transduction were found (signal proteins,
receptors, protein kinases, protein phosphatases, transcription factors and
cell adhesion protein). Interestingly, cell adhesion proteins and receptors
were particularly well represented (Table
1;
http://www.igh.cnrs.fr/equip/WebFM/).
This suggests that engrailed could act at different molecular levels
in several developmental processes.
In vitro analysis of specific Engrailed recognition motifs
A motif research analysis was performed on a subset of 107 sequences from
the 203 clones selected in the UV-X-ChIP library (see Materials and Methods).
It revealed that the most frequent motifs were a group of 49 related
octanucleotides, compiled in a position weight matrix presented in
Fig. 2A and resolved as a
`YAATYANB' consensus. This consensus sequence largely overlaps those already
described for Engrailed (Desplan et al.,
1988; Kissinger et al.,
1990
).
In order to verify that Engrailed binds to this consensus in vitro, we
performed a gel shift assay on the most represented motif `CAATTAGC', used as
a pentamer. Several retarded complexes are formed in the presence of HS-EN
protein with an affinity close to 10-9 M
(Fig. 2B). The formation of
these complexes is competed in the presence of either a single double strand
unlabelled motif `CAATTAGC' (referred as to C) or with a known specific
Engrailed binding fragment D2 (Serrano et
al., 1995). Furthermore, super-shifts of the complexes are
observed in the presence of a specific anti-Engrailed antibody 4F11
(Fig. 2B). By contrast,
addition of either a mutated form of the motif `CAGCCGGC' (referred as to Cm)
or polyhomeotic N fragment, which does not bind Engrailed
(Serrano et al., 1995
), had no
effect on the formation of the complexes. Together, these data show that
Engrailed binds specifically to this motif.
Gel shift assays were then performed on 14 Engrailed-binding fragments
isolated from the library, and the results are shown here in four cases where
the associated target genes are involved in different signaling pathways
(Fig. 3). 1A4 clone corresponds
to a genomic fragment lying 5 kb downstream of frizzled 2
(fz2), which encodes one of the wingless (wg)
receptors (Cadigan et al.,
1998). 2H10 clone corresponds to a genomic fragment lying within
hibris (hbs), which encodes a member of the immunoglobulin
superfamily involved in muscle guidance
(Artero et al., 2001
;
Dworak et al., 2001
). 1B12
clone corresponds to a genomic DNA fragment lying within the first intron
of branchless (bnl), encoding the Drosophila
homologue of the Fibroblast Growth Factor (FGF) involved in tracheal
morphogenesis (Sutherland et al.,
1996
). 2C5 clone corresponds to a genomic fragment lying in the
first intron of frazzled (fra), which encodes a netrin
receptor involved in motor axon guidance
(Kolodziej et al., 1996
).
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In each case, two sets of experiments were performed, either with the entire immunoprecipitated fragment (150 bp to 350 bp), or with a shorter 100 bp fragment, surrounding the YAATYANB motifs, previously defined in Fig. 2A. Similar results (number of complexes, affinity) were obtained for both types of probes, and the data presented in Fig. 3 correspond to the shorter 100 bp fragments.
All these DNA fragments formed retarded complexes in the presence of HS-EN protein (Fig. 3). Addition of D2 DNA was able to compete the formation of the complexes, while addition of N DNA, as expected, had no effect. Moreover, addition of 4F11 antibody super-shifted the complexes. These data show the specificity of Engrailed binding, which was also confirmed using purified Engrailed protein (data not shown). The addition of the cold DNA fragment itself allowed us to compare the affinity of Engrailed on this fragment to the affinity of the strong Engrailed-binding fragment D2 (compare lanes D2 and A in Fig. 3). The affinities are at least 10-9 M and are closely related to the affinity of the motif `CAATTAGC' (compare Fig. 2B with Fig. 3).
In conclusion, we have shown that Engrailed is able to bind specifically to these four in vivo immunoprecipitated DNA fragments, which lie close to genes involved in different developmental processes, most probably via the `YAATYANB' consensus sequence that we have identified.
The expression of potential target genes depends on
engrailed regulation in vivo
In order to discriminate among the list of putative targets, the ones
responding to engrailed regulation in vivo, we used a simple screen.
We monitored by in situ hybridization, the expression of several potential
target genes, after ectopic expression of Engrailed using the UAS-GAL4 system.
Because Engrailed can act as a repressor or an activator
(Serrano et al., 1997;
Serrano and Maschat, 1998
), we
overexpressed either the wild-type Engrailed protein (UAS-En) or a chimeric
activator form (UAS-VP16-En) (Lecourtois
et al., 2001
; Alexandre and
Vincent, 2002
), under the control of MS1096-Gal4, in
third instar wing imaginal discs (Fig.
4). We first tested this approach on ß3-tubulin,
which we have previously shown to be directly repressed by engrailed
(Serrano et al., 1997
). As
expected, overexpression of wild-type Engrailed protein led to a repression of
endogenous ß3-tubulin in the wing disc
(Fig. 4F), whereas
overexpression of the activator form of Engrailed had no detectable effect,
probably because of the strong expression of endogenous ß3
tubulin in the discs (Fig.
4D,E).
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Using this assay, we studied the expression of 14 genes that are localized close to the genomic DNA fragments isolated in the library and tested previously for their Engrailed-specific binding ability (Fig. 1B). The results are shown for four genes (frizzled2, hibris, branchless, frazzled) that are representative of the different pathways where engrailed seems to be involved (Table 1; Fig. 3). frizzled 2 expression is activated in the presence of (VP16-En) (Fig. 4H) and repressed in the presence of En (Fig. 4I) (see wild-type expression in the wing pouch for comparison, Fig. 4G). This suggests that engrailed might act as a repressor on fz2 expression. hibris is expressed along the wing margin and in the presumptive region of wing vein L3 and L4 in wild type (Fig. 4J). This expression is slightly activated in the presence of (VP16-En) (Fig. 4K), but strongly repressed when En is overexpressed (Fig. 4L), suggesting that hbs expression is regulated by engrailed in vivo. branchless is essentially expressed in a dorsal/posterior territory surrounding the wing pouch in wild type (Fig. 4M). In the presence of (VP16-En), several additional patches of bnl expression are detected within the wing pouch (Fig. 4N), whereas no activation of bnl is observed after wild type En overexpression (Fig. 4O). As expected, because MS1096 drives Gal4 expression only in the wing pouch (Fig. 4B), endogenous bnl expression outside the wing pouch is not affected (Fig. 4M-O), showing the specificity of the experiment. Finally, frazzled is slightly expressed in wild-type wing disc (Fig. 4P). This expression is activated when (VP16-En) is overexpressed (Fig. 4Q), and repressed upon En overexpression (Fig. 4R).
In conclusion, these data demonstrate that the expression of several potential target genes identified via UV-X-ChIP is modulated when engrailed is misexpressed. This test has been successfully performed on 12 genes over 14 tested (Fig. 4 and data not shown).
frizzled2 is a direct target of engrailed
regulation
Interactions between engrailed and the wingless signaling
pathway have been extensively described
(DiNardo et al., 1988;
Martinez-Arias and White,
1988
; Bejsovec and Martinez
Arias, 1991
; Heemskerk et al.,
1991
; Hatini and DiNardo,
2001
). A direct regulation of frizzled receptor
expression by engrailed has been shown
(Lecourtois et al., 2001
). In
this study, we found that the other wingless receptor gene,
frizzled2 (fz2), might also be directly regulated by
engrailed. A high-affinity Engrailed-binding fragment (1A4) was
detected in the close vicinity of the fz2 transcription unit
(Fig. 3). In wild-type embryos,
fz2 expression becomes segmentally repeated around stage 9, in two or
three rows of cells just anterior to engrailed. In stage 9
engrailed mutant embryos, fz2 expression is extended
posteriorly, being detected in 4 rows of cells
(Fig. 5A). This shows that
Engrailed acts as a repressor of fz2 expression in embryos, as has
been suggested with the previous test in the wing disc
(Fig. 4). We verified whether
the 1A4 Engrailed-binding fragment was able to drive the expression of a
reporter gene in vivo and whether it was responding to engrailed
regulation. For this purpose, this 170 bp fragment, either as a monomer or a
trimer, was cloned upstream of a GFP reporter gene and hsp70 minimal
promoter and introduced into the Drosophila genome by P
element-mediated transposition (Fig.
5B). In these transgenic lines, GFP expression was essentially
detected in the embryonic hindgut (Fig.
5C9) and in half of the larval hindgut
(Fig. 5C1). GFP is expressed in
the ventral cells of the larval hindgut that do not express
engrailed, which mimics endogenous fz2 expression
(Fig. 5D). This demonstrates
that the 1A4 DNA fragment might be a part of endogenous fz2
regulatory sequences. Overexpression of (VP16-En) fusion protein driven by
hs-Gal4 leads to ectopic GFP expression in the entire hindgut
(Fig. 5C2), but also in tissues
that do not express the transgene in wild type, such as the midgut
(Fig. 5C3,C4), the salivary
glands (Fig. 5C5,C6), and the
wing disc (Fig. 5C7,C8).
Overexpression of (VP16-En) fusion protein driven by en-Gal4 in
embryos leads to ectopic GFP expression in a striped pattern
(Fig. 5C9,C10). Such activation
does not occur with overexpression of wild-type Engrailed, confirming a
repressor role of Engrailed on fz2 expression through this 1A4
fragment (data not shown). These results show that 1A4 is able to respond to
engrailed regulation in vivo.
Finally, in order to verify that the Engrailed-binding on 1A4 was direct, we analyzed the pattern of Engrailed immunostaining on polytene chromosomes, in the transgenic 1A4-GFP line. An additional Engrailed-binding site was detected on polytene chromosomes in the locus of the transgene (25C), clearly showing a direct fixation of Engrailed on the 1A4 fragment, in vivo (Fig. 6).
|
Altogether, these data show that the 1A4 fragment that was isolated by UV-X-ChIP is a part of the fz2 regulatory regions and is able to directly respond to engrailed regulation in vivo.
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DISCUSSION |
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Using this approach, we found that potential engrailed target
genes are involved in different developmental processes, such as AP
patterning, neurogenesis, wing or tracheal development, and muscle
development. In these different pathways, we identified a number of target
genes encoding cell adhesion molecules and all types of receptors, which is in
agreement with engrailed involvement in cell-cell contact events
(Dahmann and Basler, 2000).
The identification of targets that also encode signal proteins, enzymes such
as protein kinases or phosphatases and, to a lesser extent, transcription
factors, suggest that engrailed can act at different levels on a
regulatory cascade.
Using correct markers, we notice that engrailed mutants exhibit
severe pleiotropic phenotypes. This includes defects in axon migration
(Siegler and Jia, 1999), but
also in the attachment of the muscles along the AP axis of the body
(Serrano et al., 1997
) and in
the pathfinding of the tracheal network (data not shown). These different
phenotypes could result from the abnormal setting of the compartments in
engrailed mutant embryos. The identification of potential targets
involved in these processes (Table
1) suggests rather that engrailed may be more directly
implicated in these phenotypes. This confirms the contribution of
engrailed in the orderly assembly and migration of cells during
morphogenesis and pattern formation, leading to the normal positioning of the
tissues along the AP axis.
The identification of Engrailed-binding fragments close to connectin,
18 Wheeler or eagle, which have already been defined as genetic
engrailed targets involved in neurogenesis, confirms a direct
implication of engrailed in axon guidance
(Dittrich et al., 1997;
Eldon et al., 1994
;
Siegler and Jia, 1999
).
Furthermore, we identified that the netrin frazzled receptor gene,
which is involved in motor axon guidance, might also be a direct target of
engrailed.
We isolated several Engrailed-binding fragments closely related to genes
involved in muscle development, and, in particular, genes that ensure
connections between the epidermis or PNS and the muscles. Indeed, our data
suggest strongly that the involvement of engrailed in myotube
guidance (Serrano et al.,
1997) might result, in part, from the direct regulation of
hibris (Dworak et al.,
2001
).
We also report a direct link between engrailed and tracheal
development. We indeed isolated several target genes that are involved in
directing tracheal cell migration, such as the FGF-like secreted molecule,
bnl. The tracheal system originates from placodes that consist in
part of ectodermal cells. Interestingly, bnl is not expressed in the
trachea, but in the ectodermal cells that overlie the migrating and branching
trachea, thus acting as a guidance molecule that controls tracheal cell
migration (Sutherland et al.,
1996). This result suggests a direct effect of engrailed
on tracheal guidance, which correlates with the engrailed mutant
phenotype (data not shown), although engrailed involvement in
tracheal migration has never been previously suggested.
Finally, one important and well-conserved function of engrailed
concerns its relationship with the wingless signaling pathway. The
spreading of the Wingless (Wg) signal is crucial for establishing the pattern
of differentiated cell types within tissues and organs
(DiNardo et al., 1988;
Martinez-Arias and White,
1988
; Bejsovec and Martinez
Arias, 1991
; Heemskerk et al.,
1991
; Lecourtois et al.,
2001
; Hatini and DiNardo,
2001
). This might depend in part on the level of expression of Wg
receptors. Our screen strongly suggests that the Wg receptor frizzled
2, is likely to be directly regulated by engrailed. During
hindgut morphogenesis, the morphogen dpp is repressed in the dorsal
engrailed expressing cells, but is activated in the opposite ventral
cells and it has been suggested that wg might be responsible for this
activation (Takashima and Murakami,
2001
). We show that fz2 is only expressed in the ventral
cells of the larval hindgut. Furthermore, we show that the 1A4 genomic
fragment, isolated in the library, was directly bound by Engrailed in vivo and
was effective in driving the expression of a GFP reporter gene in these
fz2-expressing cells. Our results strongly suggest that 1A4 is
responsible for the repression of fz2 by engrailed in the
dorsal cells of the hindgut, which validates the criteria of proximity to
assign a gene to one immunoprecipitated DNA fragment. The results obtained for
1A4 and in two other cases (data not shown) demonstrate that this approach
allowed us to identify genomic fragments that are functional Engrailed-binding
sites in vivo.
As shown in this report, the UV-X-ChIP technique, when associated with sequencing, provides a means to enable the rapid collection of a large data set of high-affinity binding sites used by a transcription factor during development. This method is general enough to be used to identify binding sites and targets for other transcription factors (F. G. and F. M., unpublished). Among the 203 sequences that localize within unique genomic regions, only 40 were found two or three times, indicating a low level of redundancy. This suggests that Engrailed is able to bind to a large number of genomic sites. Therefore, a more exhaustive genome-wide localization analysis should rather combine the same chromatin immunoprecipitation procedure with genomic DNA microarrays, which are not yet available in Drosophila.
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ACKNOWLEDGMENTS |
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