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First published online 8 November 2006
doi: 10.1242/dev.02689
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Department of Biology, New York University, 100 Washington Square East, New York, NY 10003, USA.
* Author for correspondence (e-mail: chris.rushlow{at}nyu.edu)
Accepted 10 October 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Dpp target gene, Dpp gradient, Zen, Feed-forward, Drosophila
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ashe and Briscoe, 2006). A
particular level of morphogen acts to elicit a specific expression profile
that eventually leads to a specific cell behavior. Many signaling molecules
have been shown to act as morphogens, including members of the Hedgehog,
Wingless and TGFß families of secreted molecules (reviewed by
Gurdon and Bourillot, 2001).
For example, gradients of Bone morphogenetic proteins (Bmps) act in
Xenopus (Wilson et al.,
1997) and Drosophila embryonic ectoderm patterning
(Ferguson and Anderson, 1992;
Wharton et al., 1993) and
Drosophila adult wing formation
(Lecuit et al., 1996;
Nellen et al., 1996). How
these gradients are interpreted by receiving cells is not fully understood.
The simplest mechanism is a direct readout of the gradient after its
conversion into a gradient of intracellular transcription factors
(Wilson et al., 1997). This
has been demonstrated in the Drosophila embryo for the Bmp ligand
Decapentaplegic (Dpp) and its transducing transcription factor, phosphorylated
Mad (Mad-P). The graded distribution of Mad-P
(Dorfman and Shilo, 2001;
Rushlow et al., 2001;
Sutherland et al., 2003) is
very similar to that of receptor-bound Dpp
(Shimmi et al., 2005;
Wang and Ferguson, 2005).
Early in cellular blastoderm formation, receptor-bound Dpp and Mad-P
are distributed at low levels in a broad dorsally localized domain, but soon a
steep stepwise gradient emerges. By the end of cellularization, high levels
are present in a five- to six-cell-wide strip of cells along the dorsal
midline, while lower levels are detected in three to four cells to either side
of the strip.
Cells respond to different levels of Mad-P by activating target
genes in nested domains that correlate with different concentrations of the
Dpp/Mad-P gradient (reviewed by
Ashe and Briscoe, 2006). For
example, Race (Ance - Flybase)
(Tatei et al., 1995) and
hindsight (hnt) [pebbled (peb) - Flybase]
(Ashe et al., 2000) are
activated in the region of high-level Mad-P. They define the highest
threshold of the Mad-P gradient and are referred to as Threshold Type
I genes (Ashe et al., 2000).
u-shaped (ush)
(Jazwinska et al., 1999) and
tailup (tup) (Ashe et
al., 2000) are expressed in a broader domain of 12 to 14 cells
comprising high and lower levels of Mad-P and are referred to as Type
II genes. pannier (pnr) extends more laterally into the
region in which Mad-P is undetectable by current techniques,
encompassing a domain of 36 to 40 cells
(Jazwinska et al., 1999), and
represents the Type III genes.
By what mechanism do these target genes respond differentially to the
Mad-P gradient? The classic paradigm of how threshold responses are
determined was established years ago from studies on the Drosophila
morphogens Bicoid (Driever et al.,
1989) and Dorsal (Jiang and
Levine, 1993), and is based on the relative affinities of
morphogen-binding sites in target-gene enhancers. Target genes activated in
regions in which low concentrations of morphogen are present contain
high-affinity binding sites in their enhancers. Targets activated in regions
of high-level morphogen contain low-affinity binding sites, but if those sites
are changed to high-affinity sites, activation can now occur in regions of
low-level morphogen (Driever et al.,
1989; Jiang and Levine,
1993). Although this mechanism has been established for some
Dorsal and Bicoid targets, it may not be sufficient to explain the
differential regulation of all their targets, or of Dpp targets. In fact, a
recent study on several Bicoid target genes using bioinformatics tools
suggested that the affinity model is insufficient to explain how threshold
borders are determined (Ochoa-Espinosa et
al., 2005).
Studies on the Dpp target Race demonstrated that in addition to
the relative-affinity mechanism, a combinatorial mechanism of interacting
activators is involved in establishing transcription limits of Dpp targets.
Support for the relative-affinity model comes from a study by Wharton et al.
(Wharton et al., 2004). The
Race enhancer contains several Smad sites, one of which has a
relatively high affinity to Smads compared with the others. When two
low-affinity sites were changed into higher affinity sites in a
Race-lacZ reporter gene, the expression border expanded into
regions containing low levels of Mad-P. However, Xu et al.
(Xu et al., 2005) showed that
a combinatorial mechanism is also utilized in which Dpp, via the Smads, and
Zerknüllt (Zen), a homeodomain protein
(Doyle et al., 1986), act
together to activate Race. Zen-binding sites lie in close proximity
to Smad sites in the enhancer, and both types of sites are required for normal
activation of the Race-lacZ reporter
(Xu et al., 2005).
Furthermore, as Zen itself is a target gene of high-level Dpp in the late
blastoderm stage (Rushlow et al.,
2001), Race regulation represents a feed-forward
regulatory circuit, in which one regulator, Dpp, activates a second regulator,
Zen, and then both activate a common target gene, Race. It was
proposed that all Type I Dpp targets are regulated in a similar way
(Xu et al., 2005).
Here we investigate the mechanism by which the Dpp target gene C15 is regulated. C15 is expressed along the dorsal midline in a five- to ten-cell-wide stripe, the lateral border of which lies within the region of intermediate-level Mad-P. Surprisingly, we found that the relative affinity of Smad-binding sites in the C15 enhancer does not play a major role in establishing this domain. Instead, combinatorial regulation involving Smad/Zen feed-forward circuitry, the relative numbers of Smad- and Zen-binding sites, and a repression mechanism is important for the C15 pattern of expression.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
sogYS06 is a null allele of sog
(Francois et al., 1994)
balanced over FM7c, ftz-lacZ. zenw36 is a null allele
(Rushlow et al., 1987)
balanced over TM3, ftz-lacZ. The double heterozygous embryos,
dpphr4/+; zenw36/+,
were identified by the lack of lacZ staining in embryos derived from
a cross of the dpphr4 and zenw36
stocks. To obtain uniform early embryonic expression of UAS-zen, a
maternal GAL4 driver was used where a GAL4-VP16 fusion
protein is expressed under the control of the 
-tubulin 67C
promoter.
Bacterial expression of Zen, Mad and Medea
The GST-Zen expression plasmid contains the full-length zen coding
region (Xu et al., 2005).
GST-MadN (Kim et al.,
1997) and GST-Medea (Xu et
al., 1998) fusion proteins contain the N-terminal MH1 domains. The
expression and the purification of the recombinant proteins were carried out
as described before (Rushlow et al.,
2001).
In vitro mutagenesis and transgenic analysis
DNA fragments summarized in Fig.
2 were prepared by PCR using genomic DNA as template (Clontech)
and the Expand High Fidelity PCR System (Roche Biochemicals). Amplified
fragments were first cloned into the pCRII vector using the TOPO cloning
system (Invitrogen) and then subcloned into the pCasPeRhs43-
ßgal transformation vector to make transgenic flies. The
mutagenesis of Zen- and Mad-binding sites was performed using the PCR method
with the same mutated oligonucleotides used in the mobility shift assays (see
Figs 3 and
4 for sequences).
In vitro DNA-binding assays
DNAse I footprint analyses and electrophoretic mobility shift assays were
carried out as previously described (Kirov
et al., 1993). For Zen footprinting assays, five fragments were
generated from the 650-3 bp C15 enhancer fragment using convenient
restriction sites: I (1-63 bp), II (59-459 bp), III (59-311 bp), IV (310-652
bp) and V (457-652 bp). For the Smad mobility shift assays, 14 fragments were
isolated: A (1-127 bp), B (124-311 bp), C (310-459 bp), D (457-516 bp), E
(517-588 bp), F (585-652 bp), G (1-60 bp), H (82-156 bp), I (169-256 bp), J
(257-299 bp), K (59-156 bp), L (154-237 bp), M (300-397 bp), N (387-459 bp).
The sequences of the wild-type and mutant oligonucleotides spanning the
putative Zen- and Smad-binding sites are listed in Figs
3 and
4.
In situ hybridization and antibody staining
Wild-type, mutant and transgenic embryos were fixed, hybridized with
C15 and/or lacZ (to detect transgenes expressed from
balancer chromosomes or to detect the lacZ transgene activity)
antisense RNA probes, stained (Roche Molecular Biochemicals), and mounted in
araldite (Polysciences) or Aquamount (Polysciences) as previously described
(Rushlow et al., 2001).
Wild-type embryos were stained with anti-phospo-Smad1 antibodies as previously
described (Rushlow et al.,
2001).
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Jagla et al.,
2001). All but C15 have been shown to be required for
mesoderm tissue specification (Jagla et
al., 2001). C15 mutant flies have appendage phenotypes,
including lack of the tarsal claws
(Campbell, 2005;
Kojima et al., 2005), hence
C15 is also called clawless (cll) (Koijma et al.,
2005). No embryonic phenotypes have been described for C15 mutants,
although C15 is expressed throughout embryogenesis
(Dear and Rabbitts, 1994).
C15 is initially expressed during stage 5 in a stripe along the
dorsal midline (Fig. 1A)
(Stathopoulos et al., 2002).
The width of the stripe varies from five to ten cells
(Fig. 1A, inset); thus, at its
widest, C15 creeps into the region in which cells contain low levels
of Mad-P (Fig. 1B).
C15 is wider than Race (a Type I target;
Fig. 1C), but not as broad as
tup (a Type II target; Fig.
1D). The variability in C15 expression along the
anteroposterior (AP) axis is not atypical for dorsally expressed genes
(Fig. 1A,C,D). C15
continues to be expressed in the dorsal region throughout gastrulation, and
can be observed in the differentiated amnioserosa and a few rows of
dorsolateral cells in older embryos (data not shown).
C15 is regulated by a Smad/Zen feed-forward loop
In order to better characterize the threshold response to Dpp of C15, we
examined its expression in dpp, zen and sog mutant
backgrounds, as well as in a situation in which Zen is overexpressed
throughout the embryo. Like the Type I gene Race
(Xu et al., 2005),
C15 is absent in dpphr4 hypomorphic
(Fig. 1E) and
dppH46 amorphic mutants (data not shown),
zenw36 amorphs (Fig.
1F), and in the double heterozygote (dpphr4/+,
zenw36/+; data not shown), although C15 is normal
in the single heterozygotes (dpphr4/+ and
zenw36/+; data not shown). The levels of Mad-P
and zen in the dpp mutant backgrounds have been reported
(Rushlow et al., 2001). In
dpphr4 Mad-P is present at a very low level,
while zen is at a medium level in a broad domain that never refines.
In dppH46 mutants, Mad-P is absent and
zen disappears in early stage 5. In zen mutants,
Mad-P is mostly normal, although the peak levels of Mad-P
are slightly diminished (Wang and
Ferguson, 2005) (C.R., unpublished). These results indicate that,
like Race, C15 requires relatively high levels of both Dpp and Zen
(Xu et al., 2005).
By contrast, C15 behaved differently from Race in
sog mutants. C15 is strongly expressed in a broad dorsal
domain comprising the middle body region
(Fig. 1G), whereas
Race expression is spotty at best
(Ashe et al., 2000;
Xu et al., 2005).
Mad-P does not form a gradient in sog mutants and never
reaches peak levels, but instead remains at a medium level throughout the
dorsal half of the embryo where C15 expression is observed
(Rushlow et al., 2001;
Raftery and Sutherland, 2003).
In addition, the zen pattern does not refine into a strong dorsal
stripe but also remains broad and at medium levels
(Rushlow et al., 2001). These
levels of Mad-P and Zen cannot activate Race
(Xu et al., 2005), but are
nevertheless sufficient to activate C15
(Fig. 1G). Thus we conclude
that C15 resembles Type II genes because it requires less
Mad-P than the Type I genes for transcriptional activation.
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), and although zen RNAs are restricted to this
domain, Zen protein is detectable in a few cells lateral to the five- to
six-cell-wide stripe, but at lower levels
(Rushlow et al., 1987). This
low-level Zen could provide a critical input for the activation of
C15. We assayed embryos in which zen is expressed
ubiquitously. We previously reported that the Mad-P gradient was not
affected in such a situation (Xu et al.,
2005). Embryos carrying UAS-zen and a
maternal-GAL4 driver showed a broadened C15 pattern,
encompassing about 26 cells at its widest point
(Fig. 1H). This domain is not
as wide as pnr (32-36 cells), but it is much wider than tup
(12-14 cells). Thus, Zen is able to activate C15 ectopically;
however, this requires some specific level of Mad-P, which in the
absence of zen is not adequate. Our genetic results support the idea that
C15 is regulated by the combined activity of Smads and Zen in a
feed-forward loop.
A 650 bp enhancer in the C15 second intron drives early blastoderm expression
To study the possibility that both Smads and Zen bind to the C15
regulatory region, we first searched for an enhancer that mediates
C15 blastoderm expression. Overlapping DNA fragments comprising the
30 kb region that includes C15 and its immediately adjacent genes
were tested in transgenic reporter gene assays for early embryonic expression
by in situ hybridization with lacZ RNA probes. The fragment
containing the second intron (I-2) was able to drive lacZ expression
in a C15-like dorsal stripe pattern
(Fig. 2B).
The I-2 fragment was divided into five smaller overlapping fragments and subjected to the same analysis (results are summarized in Fig. 2A; blue lines denote lacZ expression). Two fragments, I-2-4 and I-2-5, were able to drive blastoderm expression. I-2-5-lacZ embryos showed a C15-like dorsal stripe (data not shown), however, I-2-4-lacZ embryos showed a broader than normal dorsal stripe, especially in the middle body region (Fig. 2C), implying that the I-2-4 fragment is missing a negative regulatory element that is present in the I-2 and I-2-5 fragments.
To further delineate the early enhancer, several smaller constructs were tested (Fig. 2A). Of these, only the 650-3 (650 bp in length) and the 350-2 (about 350 bp and included within the 650-3) fragments were able to drive a lacZ pattern similar to the endogenous C15 pattern (Fig. 2D,E, respectively), although the 350-2-lacZ pattern was weaker in intensity. Both of these constructs contain sequences from I-2-5 that are not present in I-2-4, and thus harbor the putative negative element in I-2-5. Importantly, both the 650-3-lacZ and the smaller 350-2-lacZ were able to drive a C15-like dorsal stripe. Furthermore, when crossed into a sog mutant background, this pattern broadened (data not shown) like endogenous C15 in a sog mutant. These results confirm that the minimal sequences necessary for the qualitative C15 response to the Mad-P gradient is contained within the 350 bp minimal enhancer fragment. However, for a full quantitative response, additional sequences present in the full 650 bp enhancer are necessary.
The C15 enhancer contains multiple Zen and Smad sites
We used in vitro DNA-binding assays to search for Zen- and Smad-binding
sites in the 650 bp C15 enhancer. To identify Zen-binding sites, we
performed DNAse I protection (footprinting) assays with five overlapping DNA
fragments that span the 650 bp enhancer region and recombinant GST fused Zen
(GST-Zen) protein (Fig. 3A).
Protection was observed at two distinct regions in fragment IV, which we
designated Z1 and Z2, and at one region in fragment V, Z3
(Fig. 3B and C). The Z1 and Z3
sites contain an ATTA homeodomain core recognition sequence
(Kissinger et al., 1990), and
Z2 contains an AT-rich sequence, AAATTGATTT, that is similar to
the AT-rich homeodomain-binding consensus sequence, TCAATTAAAT
(Desplan et al., 1988;
Hoey and Levine, 1988). Z3
also contains an AT-rich sequence AATTTATTGTT (reverse strand
from footprint) that is very similar to the one in Z2 (identical for the seven
nucleotides in bold type). Gel shift assays with oligonucleotides spanning the
AT-rich sequences in Z1, Z2 and Z3 (Fig.
3E, putative Zen-binding sites in red) and oligonucleotides with
mutations in the AT-rich sequences (Z1m, Z2m,
Z3m; mutated nucleotides in green) further confirmed the specific
binding of Zen to these AT-rich sequences. Strong binding was observed with
the wild-type oligonucleotides (Fig.
3D, lanes 2, 6 and 10), but binding was dramatically reduced when
the AT-rich sequences were mutated (Fig.
3D, lanes 4, 8 and 12). Therefore the AT sequences in Z1, Z2 and
Z3 (two sites) represent the Zen-binding sites in the C15 enhancer.
All of these Zen-binding sites are contained in the 350-2 C15 minimal
enhancer.
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;
Xu et al., 1998). The results
are summarized in Fig. 4A
(bound fragments are denoted in blue; gels are not shown). Mad and Medea were
able to bind the same fragments, although with slightly different intensities
(data not shown).
To identify the specific sequences responsible for Smad binding, we
searched the DNA sequence of the bound fragments for the presence of GC-rich
sites characterized by clusters of GCCG, GGCG, GGCA or, more generally, GNCN,
and the Drosophila Mad consensus sequence (DSC) GCCGC[C/G]G[C/A]
found in many Dpp-responding elements (Kim
et al., 1997; Xu et al.,
1998). We searched also for the Smad-binding element AGAC (SBE or
Smad box) (Shi et al., 1998),
which is included within the reported Medea consensus site CAGACT
(Pyrowolakis et al., 2004).
Fragment A, which overlaps with fragments H and K, contains two clusters of
GNCNs (referred to as M1 and M2 in Fig.
4B, putative binding sequences in blue). Fragment B, which
overlaps with H, K and I, also contains two clusters of GNCNs (M3 and M4).
Fragment C, which overlaps with fragment M, contains two notable regions
(referred to as M5 and M6 in Fig.
4B), each with a GC-rich region (with a GNCN), an SBE and a DSC.
Fragment F contains one GC-rich region (called M7). Oligonucleotides
containing these putative Smad sites all formed complexes of varying strengths
with GST-Mad protein in mobility shift assays
(Fig. 4C). A count of the
putative Smad sites yielded between 15 and 20 (Smad-binding sites summarized
in Fig. 4E as blue ovals).
The strong binding observed with the M2 and M5 oligonucleotides (Fig. 4C, lanes 6 and 15) could be due to the presence of multiple sites on each oligonucleotide recognized by Mad proteins, which increase the total fraction of probe bound by GST-Mad in the gel shift assay. This also increases the chance of forming multiple band shifts. For example, in Fig. 4C, lane 15, higher order complexes are observed due to the binding of more than one protein molecule to an oligonucleotide. Alternatively, one of the binding sites in the oligonucleotide might have high affinity for GST-Mad and thus contribute to the bulk of the binding. In order to determine if this is the case, M2 and M5 oligonucleotides containing mutations in the Smad recognition sequences were used in mobility shift assays (listed in Fig. 4B, mutations in green). For M2, mutation of GC1, GC2, GC3 and GC4 had variable effects (Fig. 4D, lanes 2-5), with mutation of GC4 reducing binding more significantly than the others (lane 5). However, only when all sites were mutated was M2 binding abolished (lane 6). For M5, it appears that all three sites (GC, SBE and DSC) contribute to Smad binding, although mutation of the GC-rich sequence led to the strongest decrease in binding (lane 8), while mutation of the SBE (lane 9) and each of the half sites of the DSC (ds1 and 2, lanes 10 and 11) had moderate effects. Again, only when all sites were mutated (M1m) was binding completely abolished (lane 12). We can conclude that, although the single binding sites in the M2 and M5 clusters contribute differently to the total binding, no one site is essential, and therefore the degree of binding is mostly a result of cumulative action.
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The results from the DNA-binding assays indicate that there are several
clusters of Smad-binding sites in the C15 650 bp enhancer (summarized
in Fig. 4E, blue ovals).
Furthermore, two of the Smad site clusters are in close proximity to two of
the Zen sites (red ovals). The M6 oligonucleotide sequence overlaps with the
Z1 sequence by seven bases (see Fig.
4E), and the ATTA core site is 20 bases from the DSC in M6. M7 and
Z3 completely overlap, and the ATTA core site is immediately adjacent to the
GC-rich sequence (see M7 sequence in Fig.
4B). This close proximity of Smad and Zen sites is similar to the
situation in the Race enhancer, where Zen sites lie on each side of a
cluster of Smad sites (Wharton et al.,
2004; Xu et al.,
2005), although the structure of the two enhancers differs with
respect to the number of Smad site clusters and their relative strength of
binding. Comparison of the M5 cluster with a 42 bp oligonucleotide (R42 in
Fig. 4B) that contains the
highest affinity Smad site cluster in the Race enhancer
(Wharton et al., 2004) showed
that the M5 cluster has a much higher binding capability than R42
(Fig. 4C, compare lanes 13-15
with 22-24). Whether high-affinity Smad clusters play a role in activating
C15 in cells with lower levels of Mad-P was tested by
examining their in vivo relevance in transgenic enhancer-reporter
experiments.
The number, rather than the affinity, of Smad sites is important for C15 expression
As the 650 bp enhancer drives stronger expression than the 350-2 enhancer
(Fig. 2D,E), we tested mutant
binding site combinations in the context of the 650-lacZ reporter
gene. We focused mostly on the Smad sites in the 350-2 region (M5, M6, M7), as
the 350-2 can drive a C15-like pattern; however, because the M2
oligonucleotide showed strong binding, it was also tested. We induced
mutations in one or more Smad site clusters in the 650-lacZ transgene
identical to those in the M2m, M5m, M6m and
M7m oligonucleotides that completely abolished DNA binding
(Fig. 4D). lacZ
expression was unaffected in embryos carrying mutations in the M2, M5 or M6
single clusters (Fig. 5A,B, and
data not shown, respectively). Embryos carrying a mutation of M7
(Fig. 5C) exhibited reduced
levels of expression, with some cells only faintly stained, particularly those
at the lateral edges. If these cells are included in the estimate of the
number of cells across the domain, then the width is very similar to that of
endogenous C15 (see arrowheads). This reduced expression could be due
to the fact that in the M7 sequence there is a Smad site adjacent to a Zen
site (Fig. 4E). It is possible
that a Smad-Zen interaction that facilitates transcriptional activation
(Xu et al., 2005) is disrupted
in the M7 mutant, and thus expression is reduced compared with wild type.
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A repression element sets the border of the C15 expression domain
We also investigated the roles of the Zen-binding sites, which all lie in
the 350 bp enhancer (Fig. 4E).
We used the Z1m, Z2m and Z3m oligonucleotides
that completely abolished DNA binding in each cluster
(Fig. 3E) to induce mutations
in one or more Zen sites in the 650-lacZ transgene. Transgenic
embryos carrying a mutation in the Z1 site, which contains a canonical ATTA
core, showed a reduced level of lacZ expression
(Fig. 5G). By contrast,
mutation of either Z2 or Z3, both of which contain the AT-rich sequence,
caused a significant expansion of lacZ into the dorsolateral region
(Fig. 5H,I), indicating that
the Z2 and Z3 sequences mediate repression of C15 in lateral regions,
and in doing so set the border of the C15 domain. If multiple
Zen-binding sites are mutated, overall expression is diminished, presumably
because there is less activation in the absence of Zen sites. Mutation of the
Z1 and Z2 sites resulted in a similar expansion as in Z2m, but the
overall expression was weaker (data not shown), and mutation of all three
sites caused a severe reduction of lacZ expression
(Fig. 5J), indicating that all
three sites are important for activation of C15. Without activation
in these embryos, it is not possible to visualize the lack of repressor
function.
We also examined embryos carrying mutations in both the Z2 site and Smad sites. We reasoned that if the expression seen in lateral regions in Z2m embryos is dependent on low levels of Mad-P, then abolishing Smad sites might abolish lateral expression. When the M5 cluster of Smad sites was removed in the Z2 background (M5Z2m; Fig. 5K), the same expanded pattern was observed as in Z2m alone, but when two Smad site clusters were removed (M2M5Z2m; Fig. 5L) the pattern narrowed, suggesting that lower levels of the Mad-P gradient in lateral regions are insufficient to activate a reporter with compromised Smad sites.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Ashe et al., 2000). The Dpp
target C15 has an expression domain with an intermediate width
between the Type I gene Race and the Type II gene tup, yet
it can be classified tentatively as a Type II gene because it has the ability
to respond to lower than peak levels of Mad-P
(Fig. 1A,G). Here we
investigated the genetic requirements and the enhancer structure of the
C15 gene in order to gain further insight into the mechanisms of its
response to the Dpp morphogen. Our results show that three major mechanisms
are involved in establishing the unique C15 domain: (1) combinatorial
action of the Dpp signal-transducing Smads and the Zen transcription factor,
which is mediated by a genetic feed-forward loop manifested by the dependence
of Zen on Dpp, and the requirement of both of them for C15
activation; (2) an enhancer structure comprising multiple clustered Smad and
Zen sites, which ensures enhancer activation in the regions of the embryo with
intermediate levels of both transcription factors; and (3) a repression
mechanism that prevents C15 expression in more lateral regions of the
embryo, thus setting its expression boundary. As a result, the C15
enhancer is able to respond to lower threshold levels of Dpp without driving
expression as broad as Type II genes.
|
), but also
regulates expression in regions of the embryo where there are intermediate
levels of Smads and Zen. This is reminiscent of the regulatory loop
established between Dorsal and its target twist in the regulation of
intermediate-level Dorsal target genes in the neuroectoderm (reviewed by
Stathopoulos and Levine,
2002), signifying that common regulatory strategies underlie the
response to different morphogens. In experiments in which repression of the
C15 enhancer was abolished, the reporter was expressed in a much
broader pattern (Fig. 5H),
suggesting that neither Smad nor Zen is limiting for C15 expression
in its normal domain. One can conclude, then, that the Smad/Zen feed-forward
loop does not determine the expression limits of C15, rather its
ability to be activated. And again, this is in contrast to the high-level
target Race, where Smads/Zen play a direct instructive role in
setting the border of expression (Xu et
al., 2005). Still, C15 cannot be expressed in more
lateral regions of the embryo where there are lower levels of Smads that allow
expression of targets such as pnr, even in the presence of large
amounts of ectopically expressed Zen (Fig.
1H), so the C15 enhancer does have its limits in
responding to Smads and Zen. The results of our DNA-binding assays further corroborated the involvement of Smads and Zen in C15 activation. There are about 15 to 20 Smad sites and three to four Zen sites in the C15 650 bp enhancer (Figs 3 and 4). A smaller enhancer of 350 bp (350-2), which contains fewer Smad sites, seven to nine, and all of the Zen sites (see Fig. 4E), also mediates a normal C15 pattern, although it is weaker (Fig. 2E). The other small enhancer construct, 350-1, which overlaps with 350-2 by 66 bp, contains plenty of Smad sites (15 to 18), but no Zen sites, and cannot mediate expression, pointing to the importance of the combined activity of Smads and Zen.
Are some of the Smad-binding sites more important for the transcriptional
activity of C15? In the context of the 650 bp enhancer, removal of
either of the high-affinity clusters (M2 or M5) had no effect on reporter
expression, and only when additional clusters were also removed was expression
lost (Fig. 5F). However,
mutation of the M7 cluster reduced reporter expression considerably
(Fig. 5C). Although M7 does not
show strong binding to Smad proteins, we noticed its close proximity to a Zen
binding site. Previously we have shown for the Race enhancer that
close clustering of Smad- and Zen-binding sites ensures their cooperativity
(Xu et al., 2005), suggesting
that such effects might be involved in the activation of C15. The
importance of the clustering of transcription factor-binding sites for their
cooperative effect was demonstrated recently for Dorsal and Twist
(Zinzen et al., 2006). Similar
to the findings reported there, our results support the idea that structural
features of the target enhancer, such as the number of modules (binding
sites), their close proximity, and the strength of interactions between them,
is more important for threshold response than binding affinities.
Setting the expression limits of C15
Results from the mutation analysis of the individual Zen-binding sites
strongly suggest that a repressor binds to the AT-rich site present in each of
the Z2 and Z3 sequences. Embryos carrying the 650-lacZ transgene with
mutated Z2 or Z3 sites show an expanded lacZ domain
(Fig. 5H,I) suggesting that in
the wild-type situation a repressor binds to the AT sites and sets the border
of the C15 domain. What is the repressor? It is not Dorsal or
Brinker, because neither binds to AT-rich sites
(Ip et al., 1999;
Rushlow et al., 2001).
Moreover, the dorsoventral (DV) border of C15 expression is normal in
brk mutant embryos (M.L., unpublished). It is more likely to be a
homeodomain protein that interacts with the AT-rich type of
homeodomain-binding site (TCAATTAAAT)
(Desplan et al., 1988;
Hoey and Levine, 1988). The
repressor may be ubiquitously present in the blastoderm embryo or localized to
the lateral region. Several new DV genes have been identified in microarray
screens for genes differentially expressed along the DV axis, and many of
these are expressed broadly in the lateral ectoderm
(Stathopoulos et al., 2002;
Biemar et al., 2006).
A model for C15 regulation
Based on our results, we propose the following model for C15
regulation. C15 is activated by the combined action of Smads and Zen
in the dorsal region of the embryo. The border of the C15 domain is
established by an unknown repressor that binds to the same AT-rich sites to
which Zen binds, and competes with Zen for binding. C15 expression
would then depend on a fine balance of the concentration of Smad/Zen
activators and the repressor. The threshold response of C15 that we
observed in wild-type and mutant embryos can be accounted for by this model.
In the wild-type embryo there is a level of Zen and Smads in the dorsolateral
cells, where the repressor is overcome and hence C15 is expressed. In
more lateral cells, the level of Zen is lower, the repressor outcompetes Zen
and C15 is not expressed. In the sog mutant embryo, where
both Zen and Smads are at medium-high levels in the entire dorsal region
(Rushlow et al., 2001), the
repressor is outcompeted and C15 is activated
(Fig. 1G). In embryos in which
Zen is overexpressed, the repressor is outcompeted and C15 is
activated in lateral regions (Fig.
1H); however, activation is not ubiquitous, strongly suggesting
that Zen acts in concert with a certain threshold of Mad-P to
activate C15. These results further support the idea that a
particular combined level of Zen and Mad-P is required for
activation. Below this combined threshold level, C15 cannot be
activated.
One prediction from this model is that it should be possible to manipulate Smad sites such that a change in the border of the C15 expression domain results. Although none of the Smad site mutant combinations showed a dramatic border change (Fig. 5A-F), we did observe changes when multiple Smad sites were mutated in the Z2 mutant background; i.e. when repressor function was compromised. Elimination of one Smad site cluster in this background had little effect and expression remained broad (Fig. 5K), as expected because mutating one Smad site cluster had no effect before. However, when two Smad site clusters were mutated in the Z2 mutant background, the C15-lacZ domain narrowed (Fig. 5L), as if there was now insufficient Smad activity in lateral regions to activate C15-lacZ.
Our results demonstrate that the threshold response to the Dpp gradient involves a combinatorial mechanism that includes feed-forward loops and repressor/activator competition. Further studies on additional target genes is required to determine whether any Dpp target genes utilize binding-site affinity as a means to establish threshold responses.
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ACKNOWLEDGMENTS |
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