Biozentrum der Universität Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland
Authors for correspondence
(markus.affolter{at}unibas.ch
and
merabet{at}ibdm.univ-mrs.fr)
Accepted 31 January 2005
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SUMMARY |
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Key words: Transcription, Enhancer, Hox proteins, Binding selectivity, Gene regulation
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Introduction |
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This paradox has been partially solved by the finding that Hox proteins
bind to DNA in association with TALE (three amino acid extension) homeodomain
proteins: Extradenticle (Exd; Pbx proteins in vertebrates) and Homothorax
(Hth; Meis/Prep proteins in vertebrates)
(Pai et al., 1998;
Peifer and Wieschaus, 1990
;
Rauskolb et al., 1993
;
Rieckhof et al., 1997
). In
Drosophila, the formation of the trimeric complex relies on Hox/Exd
and on Exd/Hth interactions. These protein contacts raise DNA-binding affinity
of Hox proteins. In addition, as each partner of the complex contacts DNA, the
nucleotide site is larger than the Hox monomer site, leading to an increased
specificity in target site recognition
(Chan et al., 1994
;
Chan and Mann, 1996
;
Ryoo and Mann, 1999
).
One of the best characterized cis-acting elements regulated by a
Hox protein is the autoregulatory enhancer of the homeotic gene
labial (lab) (Grieder et
al., 1997; Marty et al.,
2001
; Tremml and Bienz,
1992
). The full-length enhancer recapitulates endodermal
lab expression from stage 12 onwards and integrates signalling and
Hox inputs through different modules. The Hox responsive element (HRE) has
been narrowed down to a 47 bp sequence. It is composed of a unique binding
site for Lab and its co-factors Exd and Hth
(Marty et al., 2001
;
Ryoo et al., 1999
), and
drives, on its own, expression in a subset of lab-expressing
cells.
An important characteristic of the lab enhancer is its functional
conservation in worms, flies and mouse. Trans-species analysis of the
autoregulatory enhancers of the mouse Hoxb1 or of the worm
ceh-13 genes have shown that these elements behaved like the
lab gene in Drosophila midgut endoderm; suggesting that
molecular mechanisms underlying lab class gene autoregulation are
evolutionary conserved (Popperl et al.,
1995; Streit et al.,
2002
). This conclusion is further supported by the findings that
all these enhancers harbour the same Lab/Exd-binding site composed of the
consensus sequence TGATGGAT(T/G)G.
The lab HRE served as a paradigm to establish the Hox-binding
selectivity model, a model that explains how distinct Hox proteins in a
complex with the same co-factor, Exd, reach distinct DNA-binding properties.
Analysis of protein-DNA contacts indicated that Labial and Extradenticle bind
the TGAT[GG]ATGG sequence in a head-to-tail orientation
(Chan and Mann, 1996), with Exd
and Lab contacting, respectively, the 5' TGATGG and 3'GGATGG
nucleotides; a prediction that was later confirmed by crystallographic studies
(Piper et al., 1999
). The
first GG nucleotides of the Lab half site are contacted by the N-terminal arm
of the HD, while the other nucleotides contact the third helix of the HD. Most
importantly, it was shown that mutation of the two central nucleotides of this
heterodimer site (enclosed inside brackets) from GG to TA was sufficient to
change the Hox protein in the complex; from Lab to Ultrabithorax or to
Deformed (Chan and Mann, 1996
;
Chan et al., 1997
). These
observations, together with similar experiments carried out on another
enhancer (Ryoo and Mann,
1999
), led to the proposal of the `DNA-binding selectivity model',
which defines a prototypical Hox/Exd-binding site composed of the TGAT[NN]ATNN
sequence. In this model, the two central NN nucleotides are instructive with
regard to the Hox protein that would associate with Exd: subtle differences in
DNA sequences will thus result in selecting different Hox/Exd complexes.
According to the Hox DNA-binding selectivity model, we thought that target genes for Lab should be found in close vicinity to the consensus TGAT[GG]ATGG sequence, that should be further linked to a CTGTCA Hth-binding site. We thus screened the Drosophila genome for such sites, and tested neighbouring transcripts for expression and regulation by Lab. Surprisingly, the approach led to the identification of a single novel Lab target gene, whose regulation yielded unexpected findings with regard to the Hox DNA-binding selectivity model.
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Materials and methods |
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In situ and antibody staining
Embryo collection, in situ hybridisation and immunodetection to whole
embryos were performed according to standard procedures. Digoxigenin-labelled
lab and CG11339 antisense RNA probes were generated
according to the manufacturer's protocol (Boehringer-Mannheim) with SP6
promoters. The anti-ß-Gal antibody was produced in mouse (Promega), and
the Lab antibody was produced in rabbit and affinity purified (U. Nussbaumer
and M. Affolter, unpublished). Secondary antibodies were conjugated with
horseradish peroxidase (AB kit) or FITC/RITC fluorochromes (Jackson). For Lab
fluorescent staining, the signal was amplified with the aid of a Tyramid
Signal Amplification kit (NEN life sciences).
Electrophoretic mobility shift assays (EMSAs)
Proteins for EMSA were produced with the TNT T7-coupled in vitro
transcription/translation system (Promega). EMSAs were performed in 20 µl
as described (Popperl et al.,
1995). The Lab protein used in this study was either full-length
(EST clone RE63854 obtained from the UK HGMP Resource Centre, cloned into
PcDNA3: Fig. 4A, lanes 3-8,
11-13, 16-18, 24-26; and Fig.
5B) or from amino acid 158 to its C terminus
(Fig. 4A, lanes 21-23, and
Fig. 4B, lanes 3-5;
(Chan and Mann, 1996
). His-Exd
was full length (a gift from Richard Mann:
Fig. 4A and
Fig. 5B) or from residue 1 to
323 (Fig. 4B)
(Chan et al., 1997
). The
His-Hth construct (Ryoo et al.,
1999
) included amino acids 59 to the C terminus subcloned into
pET14b (Novagen). The proboscipedia
(Cribbs et al., 1992
), Sex
combs reduced (LeMotte et al.,
1989
), Deformed (Lin
and McGinnis, 1992
), Antennapedia
(Schneuwly et al., 1987
),
abdominalA (Merabet et al.,
2003
) and AbdominalB
(Celniker et al., 1989
) cDNAs
were full length cloned in PcDNA3-expressing vector (Invitrogen). Ubx
cDNA was full length in T7pLink vector
(Galant and Carroll, 2002
).
Each Hox cDNA was sequenced and analysed for protein expression by band shift
experiments on control oligonucleotides containing a consensus
Hox/Exd/Hth-binding site (Gebelein et al.,
2002
).
|
|
Fly stocks and transformants
Transformant lines were generated by standard procedures. All
Drosophila reporter genes were generated by standard cloning
procedures, and genomic inserts were cloned into the Asp718 site of the
nuclear lacZ encoding P element vector pCß (a gift of Konrad
Basler). Original genomic inserts and mutations of the 150 bp fragment
EVIII were generated using a PCR-based approach with 5' and
3' primers coupled to a Asp718 site. For each reporter construct, the
lacZ expression pattern was determined for several independent
transformant lines. In each case, the majority of transformants of a given
construct showed identical expression patterns. For the analysis of expression
in mutant backgrounds, the following alleles were used:
labvd1 (Diederich et
al., 1989), hthP2 (kindly provided by R. Mann)
and exdYO12 [which was used to generate female germline
mosaics as described by Rauskolb et al.
(Rauskolb et al., 1993
)].
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Results |
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CG11339is regulated by Lab, Exd and Hth
Two cDNAs, LP8211 and CG11339, which map in the vicinity
of the TGATGGATGG(N)(14)CTGTCA sequence at 100B4-B5, were recovered from EST
clones. Both correspond to the same transcription unit; LP8211
containing additional 5' end sequences that include the
Lab/Exd/Hth-binding site (Fig.
2A). In situ hybridization with antisense probes derived from
LP8211 or CG11339 cDNAs were identical (not shown).
|
|
The genomic sequences responsible for lab-dependent expression of CG11339 do not include the consensus Lab/Exd/Hth site identified by the in silico approach
To identify the enhancer responsible for lab-dependent expression
of CG11339, we first tested a 2 kb genomic fragment containing the
consensus Lab/Exd/Hth-binding site identified by our in silico approach
(fragment A: Fig. 2A). This
fragment overlaps the 3' end of the first exon and the 5' end of
the first intron of the longest isoform of CG11339
(Fig. 2A). It was cloned
upstream of a lacZ reporter gene and transgenic lines for this
construct were obtained by P-element mediated transformation. Surprisingly,
this DNA element did not display any enhancer activity
(Fig. 2B). Among several other
genomic fragments tested (not shown), one was able to drive a lacZ
expression profile similar to CG11339. This 2 kb genomic element maps
200 bp upstream of the most 5' sequences of CG11339 (fragment
B: Fig. 2A and 2B). Analysis of
three subelements (fragments C-E: Fig.
2A,B) allowed us to restrict the enhancer region of
CG11339 to a 769 bp fragment (fragment E).
The fragment E drives lacZ expression in midgut endoderm and in the head, but not in the visceral mesoderm (Fig. 2B). Moreover, activity of this enhancer displayed the same genetic requirements as expression of CG11339, as it was inactive in embryos homozygous mutant for lab and hth (not shown). To further narrow down the sequence responsible for expression of CG11339 in the endoderm, eight overlapping sub-elements of the E enhancer were analysed (elements EI to EVIII: Fig. 2A). Three elements reproduced the lacZ expression profile of the E enhancer (fragments EII, EIII and EVIII; Fig. 2B), although these enhancers drive a somewhat broader expression in the endoderm, and in scattered cells in the amnioserosa. As positive enhancers EII and EIII include EVIII, we concluded that the 150 bp enhancer EVIII (and not the original 2 kb enhancer that included the consensus Lab/Exd/Hth site identified by in silico approach) bears the sequences responsible for lab-dependent expression of CG11339.
A sequence divergent from the consensus Lab/Exd/Hth-binding site is responsible for activity of the EVIII enhancer in the endoderm
Examination of sequence of the EVIII enhancer failed to identify a
consensus Lab/Exd/Hth-binding site. We thus considered that the Lab/Exd/Hth
complex might recognise a divergent sequence, and searched for presence of a
general Hox/Exd TGAT[NN]ATNN motif, with the central NN nucleotides distinct
from the ones that have so far been shown to confer Lab recruitment. One such
site was identified (TGAT[CA]ATTA; Fig.
3A). This site is separated by three nucleotides from a sequence
CTGACT, which differs by two nucleotides (underlined)
from the Hth consensus binding site CTGTCA. We also searched for Exd and Hox
half sites that would be arranged in a non conventional orientation. This led
to the identification of a second Hox/Exd-like binding sequence, ATTATTGATCG,
with the 5' end of the Exd-binding site overlapping the 5' end of
the Hox-binding site (Fig. 3A).
This atypical Hox/Exd sequence is not flanked by a putative Hth-binding site.
Alignment of Drosophila melanogaster and Drosophila
pseudoobscura sequences further showed that both sites are located within
two blocks of conserved sequence, suggesting that they might be functionally
important for CG11339 regulation.
|
The Lab/Exd/Hth complex binds the EVIII enhancer to a sequence divergent from the consensus binding site
To test for direct binding of Lab and its co-factors Exd and Hth on the
EVIII enhancer, the conserved element bearing the functional
divergent Lab/Exd/Hth-binding site was used as oligonucleotide for band shift
experiments (underlined by blue broken line in
Fig. 3A). We found that Lab
alone (Fig. 4A, lane 3), with
Exd (Fig. 4A, lane 4), or with
Exd and Hth (Fig. 4A, lane 5),
binds on EVIII. Binding specificity was confirmed by the addition of
a polyclonal Lab antibody, that inhibited Lab binding
(Fig. 4A, lane 6), but also
Lab/Exd (Fig. 4A, lane 7) and
Lab/Exd/Hth (Fig. 4B, lane 8)
complex formation.
To verify that the putative Hox, Exd and Hth sites identified within enhancer EVIII are responsible for assembling a Lab/Exd/Hth complex, we mutated independently the core sequences expected to bind each of these factors. Mutation of the Hox half site resulted in the loss of Lab (Fig. 4A, lane 11), as well as Lab/Exd (Fig. 4A, lane 12) and Lab/Exd/Hth (Fig. 4A, lane 13) binding. Mutation of the Exd half site did not impair Lab monomer binding (Fig. 4A, lane 16), but abolished the formation of Lab/Exd (Fig. 4A, lane 17) and Lab/Exd/Hth (Fig. 4A, lane 18) complexes. Finally, mutation of the Hth core site resulted in the selective loss of the Lab/Exd/Hth triple complex, while not affecting binding of Lab and Lab/Exd (not shown).
A comparison between the EVIII and the lab48/95 or
repeat3 enhancers highlights important differences in the Lab
DNA-binding properties. First, Lab binds as a monomer to EVIII, while
it can not do so on repeat3 (Chan
and Mann, 1996) or lab48/95
(Grieder et al., 1997
).
Second, although Lab and Exd cooperatively bind on these two enhancers, no
cooperative binding was observed on EVIII. The previous reports that
characterized Lab DNA-binding properties used truncated Lab and Exd proteins.
This however does not account for the observed differences, as full-length Lab
still does not bind as a monomer (compare lanes 21 and 24 in
Fig. 4A), and synergises with
Exd for complex formation on lab48/95 (compare lanes 22 and 25 in
Fig. 4A). Further support for
the idea that the distinct binding behaviour of Lab relies on intrinsic
properties of the EVIII site comes from the observation that
truncated Lab also binds EVIII as a monomer
(Fig. 4B, lane 3) and does not
cooperate with truncated Exd for DNA binding
(Fig. 4B, lane 4).
To assess to what extent EVIII is selective in assembling Hox/Exd or Hox/Exd/Hth complexes, band shift experiments with all other Drosophila Hox proteins were performed. The results demonstrate a high degree of selectivity: dimeric Hox/Exd or trimeric Hox/Exd/Hth complexes only form with Lab (Fig. 4B, lanes 4 and 5), while the other Hox proteins only bind to EVIII as monomers and with distinct binding affinities (Fig. 4B, lane 6-26). We concluded from these experiments that the Lab/Exd/Hth triple complex specifically recognizes the TGAT[CA]ATTACAGCTGACT sequence of EVIII, which strongly diverges from the established consensus TGAT[GG]AT(T/G)G (N)(1-40) CTGTCA sequence.
Sequence requirements for Lab, Lab/Exd and Lab/Exd/Hth binding to EVIII
As the Lab binding site of EVIII was strongly different from the
repeat3 and lab48/95 sequences, we investigated its
nucleotide requirements for the Lab/Exd assembly. For this purpose, we
analyzed the effects of several mutations (listed in
Fig. 5A) by band shift
experiments. In the canonical Hox/Exd-binding site, the proteins contact
nucleotide from the same DNA strand in a given orientation. Mutations
rev1 (Fig. 5B, lanes
8-10) and rev2 (not shown), that respectively alter the orientation
or strand location of the Lab-binding site abolish Lab binding, alone or as
part of a dimeric or trimeric complexes. We next generated mutations altering
the two central nucleotides CA of the TGAT[CA]ATTA Lab/Exd-binding site
present in EVIII. We observed that the effect of the mutation was
dependent of the identity switch. When these nucleotides are changed to TT
(oligonucleotide C5A6>T5T6), no
binding of Lab (Fig. 5B, lane
13), Lab/Exd (Fig. 5B, lane 14)
or Lab/Exd/Hth is observed (Fig.
5B, lane 15). By contrast, changes to GG (oligonucleotide
C5A6>G5G6), i.e. the identity
found in repeat3 and lab cis-regulatory sequences, decrease
Lab monomer binding (Fig. 5B,
lane 18), whereas Lab/Exd (Fig.
5B, lane 19) and Lab/Exd/Hth
(Fig. 5B, lane 20) complexes
form more efficiently. Finally introducing a T in place of a C at position 5
(oligonucleotide C5>T5) does not impair Lab monomer
binding (Fig. 5B, lane 23), but
strongly affects the Lab/Exd (Fig.
5B, lane 24) and Lab/Exd/Hth
(Fig. 5B, lane 25) complex
formation. We thus conclude that, despite important differences with binding
to repeat3 or lab48/95, the formation of the Lab/Exd complex
on EVIII also crucially depends of the identity of the two central
[NN] nucleotides, and requires similar strand location and orientation of the
Lab half site.
A peculiarity of the EVIII target sequence is that Lab binds to it as a monomer. Drastic mutation of the core Lab binding site (oligonucleotide EVIII(labmut) in Fig. 4A, and oligonucleotide A7T8>G7G8 in Fig. 5B, lanes 28-30), as well as more discrete alterations at the 3' end of this site (oligonucleotides A10>G10, T9>G9 and T9A10>G9G10: lanes 33-35, 38-40 and 43-45 in Fig. 5B, respectively) strongly reduce or alleviate Lab monomer binding. As strong reduction of Lab monomer binding also resulted from mutations at the 5' end of the Lab half site (corresponding to the positions 5 and 6), it suggests that Lab binding to EVIII both requires nucleotides at the 5' and 3' ends of the Hox core site. Most surprisingly, while a mutated oligonucleotide at the 3' end of the Lab half site forbad Lab monomer, Lab/Exd and Lab/Exd/Hth binding (oligonucleotide A10>G10: Fig. 5B, lanes 33-35), combining this change with mutations at the 5' end of the site (oligonucleotide C5A6>G5G6;A10>G10: Fig. 5A) can result in a compensatory effect, leading to Lab/Exd (Fig. 5B, lane 49) and Lab/Exd/Hth (Fig. 5B, lane 50) complex formation. On such an altered site, Lab still does not bind as a monomer (Fig. 5B, lane 48), but the efficiency of Lab/Exd and Lab/Exd/Hth complexes formation is highly increased.
Different Lab-responsive enhancers drive spatially and temporally distinct expression patterns
As different Lab-responsive enhancers are available, we compared their
spatial and temporal characteristics. With the exception of the enhancer
identified in this study, enhancers are either from the Drosophila
lab gene [enhancers lab550 and lab48/95
(Grieder et al., 1997;
Marty et al., 2001
)] or from
the mouse Hoxb1 gene [enhancer repeat3)
(Popperl et al., 1995
)].
Sequence characteristics of the Lab/Exd or Lab/Exd/Hth binding sites are shown
in Fig. 6A.
|
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Discussion |
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Although not valid for the regulation of all Hox target genes
(Capovilla et al., 1994;
Galant et al., 2002
;
Grienenberger et al., 2003
;
Li et al., 1999
), the
Hox-binding selectivity model is a useful conceptual framework for
understanding how Hox proteins, which as monomers display similar DNA-binding
properties, reach specificity in target site recognition by interacting with a
single co-factor, Exd. This model implies that distinct Hox/Exd complexes
select different binding sites, which has been well documented. First, in
vitro studies have shown that the prototypical TGAT[NN]ATNN Hox/Exd site
recruits Lab or Ubx, depending on the identity of the two central NN
nucleotides: GG selects Lab/Exd, while TT or TA recruits a Ubx/Exd complex
(Chan and Mann, 1996
). Second,
the Distalless regulatory element that mediates repression by Ubx
contains a Hox/Exd site where the two central nucleotides are TT
(Gebelein et al., 2002
).
Third, switching the identity of these two central nucleotides from GG to TA,
within the context of repeat3, leads to the recruitment of a Dfd/Exd
complex instead of Lab/Exd, and in vivo transformed the Lab-responsive
enhancer into a Dfd-responsive enhancer
(Chan et al., 1997
). Similar
DNA binding preferences were also observed with the vertebrate Hox and Pbx
homologues (Chang et al.,
1996
).
We used an in silico approach based on the Hox DNA-binding selectivity model to find novel Lab target genes. Although the approach identified 40 putative target sequences for the Lab/Exd/Hth complex, expression analysis of half of them only identified a single novel Lab target, CG11339. This suggests that sequences mediating Lab regulatory function in vivo are insufficiently well defined, which is further supported by the finding that the regulation of CG11339 did not rely on the consensus Lab/Exd/Hth-binding site used for the in silico approach, but on a strongly divergent sequence. These results have implications both with regard to the mode of Lab DNA-binding and more generally to the Hox-binding selectivity model.
Previous work proposed that Lab is very peculiar among all other Hox
proteins, in the sense that it does not bind DNA as a monomer, but do so in
association with the co-factor Exd. Mutation of the HX motif confers to Lab
the capacity to bind DNA in absence of Exd. Accordingly, it was proposed that
the HX exerts an inhibitory effect on Lab DNA binding, which is neutralized
when interaction occurs with Exd (Chan et
al., 1996). This conclusion was reached by studying the
DNA-binding properties of Lab on the mouse repeat3 enhancer. Here, we
observed that this conclusion does not hold on another target sequence, the
EVIII enhancer of CG11339, indicating that the previous
conclusion could reflect a specialisation of Lab activity with regard to its
autoregulation, rather than a general feature that distinguish the mode of Lab
DNA binding from that of other Hox proteins.
The Hox-binding selectivity model also implies that a given Hox/Exd complex
should recognize a consensus nucleotide sequence in downstream target genes,
which, owing to the lack of well characterised Hox target sequences, still
remains to be experimentally validated. We found that the sequence responsible
for Lab-mediated regulation of CG11339 is TGAT[CA]ATTA, which
diverges from the TGAT[GG]ATTG site mediating lab autoregulation, at
the two central positions that are predicted to define the choice of the Hox
protein recruited with Exd. The fact that Lab can recognize target sequence
differing at the central NN nucleotide is also observed upon mutation of these
nucleotides from GG to TA in the lab550 autoregulatory enhancer
(Grieder et al., 1997). Thus,
Lab can form a complex with Exd and activates transcription in vivo on at
least three sequences that differ with regard to the identity of the central
NN nucleotides: GG in repeat3, TA in the mutated lab
enhancer and CA in CG11339.
As altering the GG identity of the central NN nucleotides in
repeat3 to TA or TT alleviates Lab/Exd complex assembling, the
readout of the nucleotide identity at the central NN positions most probably
depends upon neighbouring nucleotides that are different in repeat3,
lab48/95 and CG11339. Examination of the three sites shows that
the Exd half sites are conserved, while the Hox half site differs at the most
3' end. In support for a role of nucleotides lying in the Hox half site
in the readout of the identity of the central NN nucleotides, we found that
loss of Lab/Exd complex assembly following mutations at the 3' end of
the Hox half site can be reversed by modifying the two central positions
(Fig. 5). This compensatory
effect might result from subtle changes in contacting helix 3 of the HD, which
in turn might modify the sequence requirement at the central NN position for
efficient Lab/Exd recruitment. The importance of the Hox half site 3'
end sequences is further supported by the observation that Scr and Dfd both
bind in vitro and act in vivo on a prototypical Hox/Exd site that shares a TA
at the central NN position, but differs in the identity of nucleotides at the
3' end of the Hox half site: GA for Dfd
(Chan et al., 1997) and CT for
Scr (Ryoo and Mann, 1999
).
Variability in the sequence and spacing of the Hth-binding site might also
influence the choice of the Hox protein that will preferentially form a
complex with Exd and Hth. In any case, our study clearly shows that one
Hox/Exd complex can recognize divergent sequences in two different regulated
target genes. Although the two central nucleotides play a crucial role in
assembling a specific Hox/Exd complex (Chan
and Mann, 1996; Chan et al.,
1997
; Ryoo and Mann,
1999
), added complexity to the Hox-binding selectivity model need
to be considered, and the nature of these two base pairs will not necessarily
predict which Hox protein will selectively bind with the co-factor Exd.
Finally, our data might also open perspectives on the mechanisms underlying
the establishment of complex and distinct transcriptional patterns downstream
of Hox genes. Hox transcription factors are usually expressed in broad
domains, yet downstream target genes are often activated or repressed only in
part of the Hox expression domain. It has previously been shown that
regulatory regions of downstream target genes integrate signalling inputs
(Grienenberger et al., 2003;
Marty et al., 2001
), which
provides additional positional information to restrict downstream target gene
activation. These observations highlight the importance of the environment of
the Hox/Exd-binding sequence in mediating transcriptionally distinct outputs.
Here, we show that Lab responsive enhancers that bear Lab/Exd-binding sites
drive distinct expression patterns, both with regard to spatial and temporal
characteristics. It suggests that in addition to environmental cues, the
identity of the Hox/Exd sites might also be instructive.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Present address: IBDM/LGPD, Case907, Parc Scientifique de Luminy, 13288,
Marseille Cedex 09, France
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