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First published online 12 September 2007
doi: 10.1242/dev.009522
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Howard Hughes Medical Institute and Laboratory of Molecular Biology, University of Wisconsin, 1525 Linden Drive, Madison, WI 53706, USA.
* Author for correspondence (e-mail: sbcarrol{at}wisc.edu)
Accepted 7 August 2007
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Hox proteins, Repression, Smad proteins, Collaboration, Combinatorial regulation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). It is
thought that the Hox proteins regulate many target genes within individual
developing body parts and cells (Pearson
et al., 2005). However, it is not clear how the Hox proteins
selectively regulate a broad spectrum of target genes, nor is it understood
how individual Hox proteins either activate or repress target gene
expression.
Two DNA-binding co-factors, Extradenticle (Exd)
(Chan et al., 1994) and
Homothorax (Hth) (Rieckhof et al.,
1997) of Drosophila, have been demonstrated to interact
with and cooperate with Hox proteins in the regulation of certain target genes
in vivo (Gebelein et al.,
2004; Mann and Affolter,
1998; Ryoo et al.,
1999). However, these two co-factors are not expressed in many
tissues, such as the appendages (Azpiazu
and Morata, 1998;
Gonzalez-Crespo and Morata,
1995; Rauskolb et al.,
1995). Furthermore, Hox-Exd-Hth complexes activate some of their
target genes but repress others. Thus, the binding of Hox-Exd-Hth complexes to
target sites is not sufficient to account for their biological activity.
Although much focus has been placed on Exd and Hth as co-factors, it has
recently been shown that certain Hox proteins can also collaborate with other
transcription factors, specifically Engrailed (En) and Sloppy paired (Slp), in
the selection of a target gene in vivo
(Gebelein et al., 2004). In
the case of Slp, collaboration occurs in the absence of a physical
interaction. Beyond these few proteins and target genes, the prevalence of
collaboration is unknown, and the diversity of collaborating factors and their
impact on Hox protein activity has not been explored.
In D. melanogaster, the Ultrabithorax (Ubx) protein is the sole
Hox protein that shapes the differentiation of the hindwing (haltere). Removal
of Ubx activity from the developing haltere results in the homeotic
transformation from hindwing to forewing (wing) morphology
(Lewis, 1978). Ubx patterns
the haltere by modulating the expression of a variety of genes in the wing
morphogenetic program (Crickmore and Mann,
2006; Weatherbee et al.,
1998).
The molecular requirements for Ubx target gene regulation are not well
understood. The simple TAAT core nucleotide sequence of the Ubx and most other
Hox binding sites is a very common motif within gene regulatory regions that
are not Hox-responsive (Ekker et al.,
1991). It is possible that the number and/or affinity of Hox
binding sites in regulatory DNA must reach some threshold to elicit a response
(Galant et al., 2002), or that
the topology of Hox binding sites in association with other transcription
factor binding sites might be critical for Hox target gene selection.
Here, we performed genetic and biochemical analyses to identify the
transcription factors and regulatory sequences required for Ubx regulation of
the spalt [sal; also known as spalt major
(salm) - FlyBase] gene, which is directly repressed by Ubx in the
haltere (Galant et al., 2002).
Surprisingly, we found that whereas the Dpp/TGF-ß pathway is required for
sal activation in the developing wing, sal is directly
repressed by a combination of Dpp signaling input and Ubx in the developing
haltere. Furthermore, we show that the close proximity of Ubx and Smad binding
sites in the sal cis-regulatory element (CRE) is critical for target
gene repression. These results, together with recent findings
(Gebelein et al., 2004),
suggest that the Hox proteins collaborate with, and might depend upon, a wide
variety of transcription factors for target gene regulation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The
primary antibodies and their dilutions were: rabbit anti-Sal (1:1000; provided
by R. Barrio) (de Celis et al.,
1996), rat anti-Brk (1:100; provided by G. Campbell)
(Campbell and Tomlinson, 1999)
and mouse anti-Ubx FP3.83 (1:20; provided by R. White)
(Kelsh et al., 1994).
Protein expression and EMSA
GST-MadN and GST-MedMH1 were purified from Escherichia coli as
described (Kim et al., 1997;
Xu et al., 1998). Full-length
Ubx1a protein was produced by in vitro transcription and translation as
described (Promega) (Galant and Carroll,
2002). Double-stranded oligonucleotide probes with GATC overhangs
at the 5' and 3' ends were end-filled with
[
-32P]dNTPs using the Klenow fragment (Roche).
Electrophoretic mobility shift assays (EMSAs) were performed using previously
reported methods with the following modifications
(Galant et al., 2002): the
conditions for binding were 20 mM HEPES pH 7.8, 50 mM KCl, 0.25 mg/mL BSA, 1
mM DTT, 4% (w/v) Ficoll. Binding reactions were incubated on ice for 30
minutes and polyacrylamide gel electrophoresis was performed at 4°C.
Reporter constructs for the sal1.1 CRE
The sequence of the sal1.1 CRE is available in GenBank (accession
AF46408712). Mutant variants of the sal1.1 CRE were created by
site-directed mutagenesis via either two-step PCR or the QuikChange Multi
Site-directed Mutagenesis Kit (Stratagene). Primer sequences are available
upon request. Mutated sal1.1 CREs were cloned into the hsp-lacZ
CaSpeR reporter plasmid (Nelson and
Laughon, 1993). At least four independent lines for each construct
were analyzed for expression level. Representative lines are included in
figures.
Phylogenetic analysis of the sal1.1 CRE
D. melanogaster sequence of the sal1.1 CRE was aligned
with D. virilis genomic sequence using BLAST and by eye. Regions of
high nucleotide conservation flanking the sal1.1 CRE were used in the
design of PCR primers. These primers were used to PCR amplify homologous
sal1.1 CREs from D. subobscura and D.
malerkotliana. Collected sal1.1 CREs were aligned using MacClade
and by eye (Maddison and Maddison,
1989). The sal1.1 CRE from D. pseudoobscura was
cloned into hsp-lacZ CaSpeR, used in P-element mediated transgenesis
and tested for expression in three independent lines.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Sturtevant et al., 1997).
sal is not expressed in the corresponding region of the haltere
imaginal disc, owing to its direct repression by Ubx
(Fig. 1A,B)
(Galant et al., 2002). Ubx may
repress sal or other target genes through a number of mechanisms. One
possibility is that Ubx blocks the binding of an activator to sal
regulatory DNA. A second possibility is that Ubx acts independently of
activators or other proteins to repress target gene expression.
Activation of sal expression in the wing imaginal disc has been
shown to require the Dpp/TGF-ß signaling pathway
(de Celis et al., 1996;
Lecuit et al., 1996;
Nellen et al., 1996). The
Mothers against Dpp (Mad) protein is the Drosophila ortholog of
Smad1/5 and is required for the transduction of Dpp signaling in the wing disc
(Kim et al., 1997;
Raftery et al., 1995;
Sekelsky et al., 1995).
Homozygous Mad mutant clones lack sal expression, indicating
that Mad is genetically required for sal activation in the wing disc
(Fig. 1E,F)
(Lecuit et al., 1996;
Marty et al., 2000).
We were therefore surprised to observe that sal was expressed in Mad mutant clones in the haltere disc (in 24% of clones). Mad is therefore required to repress sal expression in this tissue (Fig. 1I,J), and is not required for sal activation in the haltere disc. Either the perdurance of activated, phosphorylated Mad (pMad) in cells or a restricted temporal requirement for Mad activity might account for the clones in which sal is not derepressed. sal is derepressed in larger clones further from the source of Dpp signaling along the anterior-posterior compartment boundary and these cells have lower levels of pMad. sal expression in Mad mutant clones in the haltere disc could be due to either a direct requirement for Mad to repress sal or to an indirect effect of the cell-autonomous loss of Mad activity on the expression of some other repressor of sal.
In order to test whether sal expression in Mad mutant
clones could be an indirect effect, we examined the effect of loss of pMad
activity on the expression of two repressors of sal, brinker
(brk) (Barrio and de Celis,
2004) and Ubx (Galant
et al., 2002). In wild-type wing and haltere imaginal discs,
brk is expressed in cells along the lateral edges of each disc and is
repressed in the central region by the Dpp morphogen gradient emanating from
the anterior-posterior compartment boundary
(Fig. 1C,D)
(Campbell and Tomlinson, 1999;
Jazwinska et al., 1999;
Minami et al., 1999). In
Mad clones in the wing disc, brk was expressed and in turn
repressed sal (Fig.
1E,G). In the haltere disc, brk was also expressed in
Mad clones; however, it did not repress sal expression
(Fig. 1I,L). Furthermore,
Ubx expression was not altered in Mad mutant clones in the
haltere, demonstrating that the derepression of sal is not due to a
loss of Ubx expression (data not shown). Therefore, the derepression
of sal due to the loss of Mad activity is not a secondary effect on
known repressors of sal in the haltere.
It is well established that R-Smads interact with a co-Smad in target gene
regulation (Feng and Derynck,
2005). In Drosophila, Mad, an R-Smad, interacts with the
co-Smad Medea (Med), the ortholog of Smad4
(Das et al., 1998). Schnurri
(Shn) is a co-repressor known to interact in a trimeric complex with Mad and
Med (Pyrowolakis et al.,
2004). In order to test whether Med and Shn are also required to
repress sal in the haltere, we examined sal expression in
Med and shn hypomorphic clones in the haltere disc.
sal was found to be derepressed in Med hypomorphic clones
(26% of clones) and in shn hypomorphic clones (29% of clones) in the
haltere disc (Fig. 1M-P). These
results suggest that the trimeric repressor complex of Mad-Med-Shn is required
to repress sal in the haltere and raises the possibility that the
complex acts directly upon a regulatory element of the sal gene.
A Mad/Med binding site is required for sal repression in the haltere imaginal disc
The activation of sal in the wing and its repression in the
haltere are regulated by a 1.1 kb CRE, sal1.1
(Galant et al., 2002).
Previously, we have shown that sal1.1 is directly repressed by Ubx in
the haltere (Galant et al.,
2002). In order to test whether Mad/Med binds to and directly
represses the activity of the sal1.1 CRE in the haltere, we searched
for candidate Mad/Med binding sites in the sal1.1 CRE. We identified
one candidate Mad/Med binding site, M1 (5'-AGACGGGCAC-3'), which
lies between Ubx binding sites 5 and 6 in sal1.1, using binding site
prediction and electrophoretic mobility shift assays (EMSAs)
(Fig. 2A). The sequence of M1
deviates somewhat from published Mad/Med silencer consensus binding sites
(5'-AGAC-5 bp-GNCGYC-3') (Gao
et al., 2005; Pyrowolakis et
al., 2004), and Mad and Med bound with >10-fold and >25-fold
lower affinities, respectively, to the M1 site than to the bam
(Gao et al., 2005) and
brk (Pyrowolakis et al.,
2004) silencer elements (data not shown).
In order to test whether Mad/Med bound specifically to the M1 site, we introduced a series of point mutations within the M1 site and examined their effect on protein binding in vitro. Of four point mutations to the M1 site, the single mutation at position 808 reduced the binding of a Med fusion protein (GST-MedMH1) to M1 as compared with the wild-type sequence (Fig. 2B, lanes 1-4 and 5-8). The remaining three point mutations did not affect the affinity of GST-MedMH1 for the probe (Fig. 2B, lanes 9-20). These results suggest that Med might contact the sequence 5'-AGAC-3' in sal1.1 (Fig. 2A). By contrast, the four individual point mutations each decreased, but did not abolish, binding of a Mad fusion protein (GST-MadN) in vitro, with the point mutation at bp 814 having the strongest effect (Fig. 2C, lanes 25-49). The weaker effect of the individual point mutations in M1 on Mad binding affinity in vitro is likely to be due to the affinity of MadN for both 5'-AGAC-3' Smad sites and GC-rich sequence. Combining these four mutations (sal798-824 kM1) had the greatest effect on GST-MadN binding to the probe (Fig. 2C, lanes 25-29 and 50-54). This analysis of individual point mutations indicates a putative orientation for a Mad/Med compound-binding site in the sal1.1 CRE (Fig. 2A).
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Juxtaposition of Ubx and Mad/Med binding sites is required for sal repression
The Mad/Med M1 binding site and the Ubx U5 and U6 binding sites lie
adjacent to one another. This proximity raises the possibility that mutations
in one site could affect the binding of the other protein and/or that the
proteins might contact each other. We tested whether mutations in the M1 site
affected the affinity of Ubx for its neighboring sites U5 and U6 by EMSA in
vitro (Fig. 3A,B). Initially,
we examined the activity of the Ubx homeodomain (HD) and found that it bound
similarly to wild-type (Fig.
3A, lanes 2-7) and mutated
(Fig. 3A, lanes 16-21) M1
probes in EMSAs. However, we considered that the truncated Ubx-HD protein
might be less sensitive than full-length Ubx1a to mutations in sites flanking
its TAAT binding site core sequence. Therefore, we also examined the binding
of full-length Ubx1a to probes in which the M1 site was mutated. Ubx1a bound
equally well to U5 and U6 even when all four mutations were introduced into
the M1 site (Fig. 3B, lanes
12-15), but did not bind to probes when the U5 and U6 sites were mutated
(Fig. 3B, lanes 7-10).
Similarly, binding of GST-MedMH1 (Fig.
3C, lanes 1-8) and GST-MadN
(Fig. 3D, lanes 9-18) to the M1
site was unaffected when Ubx binding sites U5 and U6 were mutated. We also
tested whether the requirement in vivo for both Ubx binding sites 5 and 6 and
the Smad M1 site were equal or additive by comparing sal1.1 reporter
constructs with mutations in either the pair of Ubx binding sites or the Smad
M1 site or both. We found that the strength and pattern of derepression were
equivalent if either the pair of Ubx binding sites 5 and 6 or the Smad M1 site
or both were mutated (Fig.
4A,B; data not shown). These results indicate that both Mad/Med
and Ubx are binding to distinct sites in very close proximity and confirm that
a reduction in the binding of any of these proteins to the CRE leads to
derepression of sal in the haltere disc.
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|
) (data not shown), nor did we detect co-occupancy of
the probe by either of the Smads and Ubx together. It is certainly possible
that our failure to detect a Hox-Smad interaction or a tripartite complex was
because the conditions we tested were insufficient for the assembly of such
complexes. Because mutations that affect Smad or Ubx binding do not affect the
binding of the other protein (Fig.
3), we have no evidence that their respective binding sites
overlap and that the binding of one protein might occlude binding of the other
protein, nor do steric considerations indicate that the M1 site cannot
accommodate both proteins. We suspect that the low affinity of Smads for the
M1 site has hampered our ability to detect complexes of both proteins on DNA.
Nonetheless, the possibility remains that the Smads and Ubx bind sequentially,
but not simultaneously, to the sal CRE.
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). In
order to test whether the position of the Ubx binding sites relative to the M1
site was critical for sal repression, we added one copy of the
binding site U5 (5'-CATATTAAGA-3') to both the 5'
and 3' ends of a sal328 element (178 bp 5' and 132 bp
3' from their native positions) in which both native sites (U5 and U6)
had been mutated. The addition of two copies of U5 to the mutated
sal328 CRE (sal328kU5&6) did not restore repression to
this element (sal328kU5&6+2U5)
(Fig. 4D). Although the
sal328kU5&6+2U5 CRE, like wild-type sal328CRE, has three
natural Ubx binding sites, we conclude that Ubx does not repress gene
expression because its sites are placed too far from the collaborating Mad/Med
binding site M1. We further tested the dependence of Ubx regulation upon Ubx monomer binding site topology by attempting to impart Ubx regulation upon a naive CRE. We tested whether the vestigial boundary enhancer (vgB), which drives reporter gene expression along the dorsal-ventral compartment boundary in both the wing and haltere imaginal discs (Fig. 4E), could be specifically repressed by Ubx in the haltere. The Ubx binding site topologies tested included the addition of a cassette of four copies of Ubx binding site 5 (5'-CATATTAAGA-3') from the sal1.1 CRE to both the 5' and 3' ends of the vgB CRE. Each copy of Ubx binding site 5 was one helical turn from its neighboring Ubx binding site. The arrays of Ubx binding sites had no effect on reporter gene expression in the haltere (Fig. 4F).
These results and the functional requirement for the proximity of the Mad/Med M1 site to Ubx binding sites U5 and U6 in the sal CRE indicate that there might be selective constraints on the sequence and arrangement of the binding sites. Indeed, alignments of the orthologous regions of the sal1.1 CRE from diverse Drosophila species revealed that not only are these crucial binding sites conserved, but a region of 37 bp encompassing these three sites is perfectly conserved (Fig. 5). This is an exceptional degree of sequence conservation among the sampled taxa and is very strong additional evidence that this Mad-Med-Shn- and Ubx-responsive CRE requires the integrity and close spacing of these binding sites to be maintained for sal repression in the haltere.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Lecuit et al., 1996;
Marty et al., 2000;
Nellen et al., 1996) and
molecular studies (Barrio and de Celis,
2004) that had indicated that the Dpp pathway and Mad/Med were
involved in sal activation in the wing. We find no direct evidence
that this is the case and the fact that sal is activated in
Mad and Med clones in the haltere indicates that
sal is activated independently of Mad/Med in the flight appendages.
The requirement for Mad/Med/Shn in shaping the pattern of sal
expression in the wing appears to be indirect - the protein complex represses
the expression of brk, a repressor of sal, in cells in the
central region of the developing wing and thereby permits sal
expression (Marty et al.,
2000; Muller et al.,
2003; Pyrowolakis et al.,
2004).
The Mad-Med-Shn complex is also active within cells in the central region
of the haltere as a consequence of Dpp signaling
(Fig. 6)
(Muller et al., 2003;
Pyrowolakis et al., 2004).
However, whereas sal is expressed and the sal1.1CRE is
active in the wing, sal and the sal1.1 CRE are repressed in
the haltere. These observations raise the question of how the Mad-Med-Shn
complex selectively represses sal in the haltere but not in the wing
disc? Our results suggest that there are two key determinants in the selective
repression of sal in the haltere. The first is collaboration with
Ubx, which is expressed in the haltere and not in the wing disc. The second
key determinant might be the affinity of Mad/Med binding to the sal
CRE.
The different responses of the brk and sal genes to
Mad/Med/Shn suggests how the different affinities of proteins for binding
sites might determine how available transcriptional regulatory inputs are
integrated by CREs (Fig. 6).
Mad/Med binding to the brk CRE is of high affinity
(Pyrowolakis et al., 2004) and
apparently sufficient to impart repression, whereas that to the sal
CRE is of much lower affinity and insufficient to impart repression in the
wing. In the haltere, although Mad-Med-Shn or Ubx binding are alone
insufficient, they act together either via simultaneous or sequential
occupancy of their binding sites to repress sal.
Collaboration as a distinct mode of combinatorial regulation
The requirement for two or more regulators to act together to control gene
expression, i.e. combinatorial regulation, is fundamental to the generation of
the great diversity of gene expression patterns by a finite set of
transcription factors. Several previous studies have revealed the dual
requirement for Hox and Smad functions for the activation of a target gene
(Grieder et al., 1997;
Grienenberger et al., 2003;
Marty et al., 2001). These
studies suggested a general combinatorial mechanism for gene activation in
which apparently separate transcriptional inputs act synergistically in gene
activation and, in at least one case, the Hox response element and Dpp
response element are separable (Marty et
al., 2001). Here, however, we have observed a requirement for and
strict evolutionary conservation of the close topology of Hox and Smad binding
sites in the sal CRE. We suggest that collaboration is a distinct
mode of combinatorial regulation in which two or more regulatory proteins must
bind to nearby sites, but not necessarily to each other.
The integration of Hox and Smad inputs could work through a number of
possible mechanisms (Guss et al.,
2001; Marty et al.,
2001) in the absence of direct physical interaction. One appealing
possibility that might explain the requirement for the close proximity of
binding sites is that Ubx and Mad-Med-Shn might interact with, and could
therefore cooperatively recruit, the same co-repressor(s) for the repression
of sal. Alternatively, if Mad-Med-Shn and Ubx bind sequentially to
sal1.1, they might recruit different co-repressors and thereby
orchestrate the assembly of a co-repressor complex. A third possibility is
that because the Ubx and Mad/Med sites are embedded within a larger block of
conserved regulatory DNA sequence in the sal1.1 CRE, the binding of
other interacting transcription factors might also be involved in the
repression of sal by Ubx and Mad-Med-Shn.
The general role of collaboration in Hox target gene selection and activity regulation
These and recent results raise the question of whether collaboration is a
general feature of target gene selection by Hox proteins
(Gebelein et al., 2004). We
suggest that collaboration might be a widespread requirement for Hox function
in vivo.
Our proposal is prompted by three observations. First, Hox proteins alone
have low DNA-binding specificity (Ekker et
al., 1991). Second, some, and perhaps all, Hox proteins might act
as both repressors and activators. Third, Hox proteins regulate a great
diversity of target genes that are also regulated by other transcription
factors. In order to be such versatile regulators, it would be too great a
constraint to require that Hox proteins always interact cooperatively with the
diverse repertoire of transcription factors with which they act. Indeed, it
may be argued that too much weight has been ascribed to the cooperative
binding of Hox proteins and co-factors to DNA.
Previously, much attention has focused on Exd and Hth, which interact with
Hox proteins and bind cooperatively to DNA, thereby increasing Hox DNA-binding
selectivity (Chan et al., 1994;
Chan et al., 1997;
Mann and Carroll, 2002;
Pederson et al., 2000).
However, it was only recently shown that the binding of these complexes alone
was not sufficient to regulate target gene expression. Rather, Hox-Exd-Hth
collaborate with and require the segmentation proteins Slp and En to repress
the target gene Dll (Gebelein et
al., 2004). Here, we have shown that the Exd- and Hth-independent
target gene repression of sal requires collaboration between Ubx and
Mad-Med-Shn. Although still a tiny sample of target genes, we now have cases
of transcription factors of various structural types acting as collaborators
with Hox proteins. The picture of Hox proteins relying on dedicated
interacting co-factors such as Exd and Hth is expanding to a larger pool of
collaborating transcription factors that modulate target gene selection.
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For instance, there is no evidence that the mere binding of Hox-Exd-Hth to
a site determines the sign of Hox activity. These co-factors are involved in
both Hox target gene activation (e.g. dpp in the midgut) and target
repression (e.g. Dll in the embryonic abdomen). But, in the latter
case, En and Slp, two proteins that each harbor motifs for interaction with
the co-repressor Groucho (Alexandre and
Vincent, 2003; Andrioli et al.,
2004; Kobayashi et al.,
2003; Lee and Frasch,
2005), are required collaborators for Dll repression. The
roles of En and Slp in this instance might not be so much a matter of
facilitating Hox target selection, but rather in regulating the sign of the
output of the collaboration.
Similar to the Hox proteins, the Smads can either activate or repress
target genes (Feng and Derynck,
2005). Furthermore, it has been demonstrated that the topology of
Smad binding sites on DNA appears to be critical for determining whether a
target gene is activated or repressed. In Drosophila, the topology of
Mad and Med binding sites is critical for the recruitment of the co-repressor
Shn (Gao et al., 2005;
Pyrowolakis et al., 2004). The
recruitment of Shn was shown here to be necessary for sal repression.
These two examples suggest that the positive or negative regulatory activity
of a Hox protein depends on the context of surrounding binding sites and how
they influence the activity of collaborating factors.
The dependence of Hox proteins upon co-factors and collaborators indicates that, at the molecular level, Hox proteins are not `master' regulatory proteins that dictate how target genes behave. Rather, they exert their great influence by virtue of their simple binding specificity, broad domains of expression and versatile, collaborative properties.
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ACKNOWLEDGMENTS |
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