Download DNA-binding domain mutations in SMAD genes yield

Research article
Development and disease
4883
DNA-binding domain mutations in SMAD genes yield dominantnegative proteins or a neomorphic protein that can activate WG
target genes in Drosophila
Norma T. Takaesu1, Eric Herbig1,*, David Zhitomersky2, Michael B. O’Connor2 and Stuart J. Newfeld1,†
1
School of Life Sciences, Arizona State University, Tempe, AZ 85287-4501, USA
Howard Hughes Medical Institute, Department of Genetics, Cell Biology and Development, University of Minnesota, Minneapolis,
MN 55455, USA
2
*Present address: Molecular and Cellular Biology Program, University of Washington School of Medicine, Seattle, WA 98195, USA
†
Author for correspondence (e-mail: [email protected])
Accepted 18 August 2005
Development 132, 4883-4894
Published by The Company of Biologists 2005
doi:10.1242/dev.02048
Development
Summary
Mutations in SMAD tumor suppressor genes are involved
in approximately 140,000 new cancers in the USA each
year. At this time, how the absence of a functional SMAD
protein leads to a tumor is unknown. However, clinical and
biochemical studies suggest that all SMAD mutations are
loss-of-function mutations. One prediction of this
hypothesis is that all SMAD mutations cause tumors via a
single mechanism. To test this hypothesis, we expressed five
tumor-derived alleles of human SMAD genes and five
mutant alleles of Drosophila SMAD genes in flies. We found
that all of the DNA-binding domain mutations conferred
gain-of-function activity, thereby falsifying the hypothesis.
Furthermore, two types of gain-of-function mutation were
identified – dominant negative and neomorphic. In
numerous assays, the neomorphic allele SMAD4100T
appears to be capable of activating the expression of WG
target genes. These results imply that SMAD4100T may
Introduction
Signaling molecules of the Transforming Growth Factor-␤
(TGF␤) family regulate differentiation and proliferation in
many species. Four TGF␤ proteins have been characterized in
Drosophila and each of these has a counterpart in mammals
(Newfeld et al., 1999). For example, Decapentaplegic (DPP)
shows significant amino acid similarity to mammalian BMP2
and BMP4. In addition, the function of these proteins appears
to be conserved. In cross-species experiments, human BMP2
and BMP4 rescued dpp mutant phenotypes in Drosophila
(Padgett et al., 1993), and recombinant DPP protein induced
bone formation in mammalian cells (Sampath et al., 1993). The
functional conservation revealed by cross-species experiments
suggests that studies of TGF␤ signaling in Drosophila will
impact on our understanding of TGF␤ signaling in mammals.
In brief, DPP signal transduction begins with a complex of
transmembrane receptor serine-threonine kinases. Upon ligand
binding, one of the receptors phosphorylates the cytoplasmic
protein Mothers against DPP (MAD) (Newfeld et al., 1996).
As a result of MAD phosphorylation, the MAD-related protein
Medea (MED) is recruited to form a heteromeric complex with
induce tumor formation by a fundamentally different
mechanism from other SMAD mutations, perhaps via the
ectopic expression of WNT target genes – an oncogenic
mechanism associated with mutations in Adenomatous
Polyposis Coli. Our results are likely to have clinical
implications, because gain-of-function mutations may
cause tumors when heterozygous, and the life expectancy
of individuals with SMAD4100T is likely to be different from
those with other SMAD mutations. From a larger
perspective, our study shows that the genetic
characterization of missense mutations, particularly in
modular proteins, requires experimental verification.
Key words: Drosophila, SMAD genes, TGF␤, Tumorigenesis,
Pancreatic cancer, Colon cancer
MAD (Wisotzkey et al., 1998). The MAD/MED complex then
translocates to the nucleus and co-activates the transcription of
DPP target genes (e.g. Xu et al., 1998).
All TGF␤ family members use this mechanism, and MADrelated proteins (the SMAD family) are found in many species.
SMAD family members contain two functionally distinct and
highly conserved MAD homology (MH) domains. The aminoterminal MH1 domain appears to be responsible for
transcriptional activation, whereas the carboxy-terminal MH2
domain appears necessary for forming multi-SMAD
complexes (Lagna et al., 1996).
TGF␤1 was discovered through its anti-mitotic effects on
fibroblast cells in culture (Barnard et al., 1990). However,
TGF␤1 was unable to induce growth arrest in fibroblastderived tumors (Fynan and Reiss, 1993). Subsequently, ‘loss
of heterozygosity’ studies have shown that human SMAD
genes act as tumor suppressors in a wide variety of tissues.
Homozygous mutations in SMAD2 or SMAD4 are detected in
20% of breast, 42% of colorectal, 17% of lung and 80% of
pancreas tumors, as well as in the inherited cancer Autosomal
Dominant Juvenile Polyposis (Eppert et al., 1996; Riggins et
Development
4884 Development 132 (21)
al., 1997; Howe et al., 1998). More recently, reporter gene
assays of SMAD mutant alleles isolated from human tumors
revealed a universal inability to activate transcription at wildtype levels (Dai et al., 1998; Xu and Attisano, 2000). These
studies led to the hypothesis that all SMAD tumor mutations
are loss-of-function mutations. One prediction of this
hypothesis is that all mutations induce tumors via a single
mechanism – the inability to transduce an anti-mitotic signal
encoded by a TGF␤ family member (e.g. Massagué et al.,
2000).
However, the modular nature of SMAD proteins and our
experience with mutations in Mad (e.g. Sekelsky et al., 1995)
suggest that the situation is more complex. We propose the
alternative hypothesis that there are multiple classes of SMAD
mutation (loss of function and gain of function). A prediction
of our hypothesis is that human SMAD mutations in different
classes induce tumor formation via distinct mechanisms.
Here, we report a study, using the Gal4/UAS system, in
which we generated phenotypes for mutant alleles of Mad and
Med, as well as for mutant alleles of human SMAD2 and
SMAD4 isolated from pancreatic and colon tumors. Our study
establishes a set of principles for the transgenic
characterization of human mutant alleles. In wild-type flies, the
expression of a loss-of-function mutation does not generate a
mutant phenotype, whereas the expression of a gain-offunction mutation generates a mutant phenotype. Furthermore,
different classes of gain-of-function mutation generate
different phenotypes. Phenotypes generated by dominantnegative alleles mimic genomic loss-of-function mutations.
Phenotypes generated by neomorphic mutations are unrelated
to the wild-type function of the gene.
Materials and methods
Sequencing Mad mutants
The entire locus was amplified by PCR from heterozygous animals.
PCR products were sequenced using the Thermosequenase cycle
sequencing kit (USB). The lesion was identified by the presence of
two bases at a particular position corresponding to the mutant allele
and the wild-type allele on the balancer chromosome.
Transgene constructs
The Mad1 allele contains an A615T mutation. By using PCR (Lorson
et al., 1999), this mutation was copied into the Mad cDNA using a
pair of complementary mutant primers. The Mad1 reverse primer 5⬘CTGGACGGACGATTACTGGTCTCCCATCGC-3⬘ (the mutant
base is shown in bold) and the M13 forward primer were used to
create the 5⬘ Mad1 fragment. The Mad1 forward primer 5⬘GCGATGGGAGACCAGTAATCGTCCGTCCAG-3⬘ and the M13
reverse primer were used to create the 3⬘ Mad1 fragment. A full-length
Mad1 cDNA was produced using the M13 forward and M13 reverse
primers, and the annealed 5⬘ and 3⬘ Mad1 fragments as a template.
The Mad12 allele contains a C1601T mutation (Sekelsky et al., 1995).
The Mad12 reverse primer 5⬘-GCGGAGTATCATCGCTAGGATGTGACCTCG-3⬘ and the M13 forward primer were used to create the
5⬘ Mad12 fragment. The Mad12 forward primer 5⬘-CGAGGTCACATCCTAGCGATGATACTCCGC-3⬘ and the M13 reverse
primer were used to generate the 3⬘ Mad12 fragment. Mad mutant
cDNAs were cloned into the XbaI and KpnI sites in pUAST (Brand
and Perrimon, 1993). SMAD4 mutant cDNAs (Schutte et al., 1996)
were excised by cutting with BamHI and EcoRV, and cloned into
blunted XhoI and BglII sites of pUAST. An asymmetric SacI site was
used to check orientation. Additional SMAD mutant cDNAs (Riggins
et al., 1996) were excised by cutting with BamHI, blunting the ends
Research article
and cutting with KpnI. cDNAs were then cloned into the XbaI
(blunted) and KpnI sites in pUAST. Multiple independent fly lines
were generated for five mutants.
Drosophila genetics
Fly stocks were as described: In(2L)dpps6 and dpphr4 (St Johnston et
al., 1990); Df(2L)C28, Mad1, Mad11 and Mad12 (Sekelsky et al.,
1995); Df(3R)E40 and Med7 (Raftery et al., 1995); zw3M11 FRT101
and zw3sggD127 FRT101 (Siegfried et al., 1992); UAS.Dpp, UAS.CAArm, UAS.DN-TCF, UAS.Mad, UAS.Gbb, UAS.lacZ, 24B.Gal4,
32B.Gal4, 69B.Gal4, A9.Gal4, C765.Gal4, MS1096.Gal4, T80.Gal4,
ap.Gal4, dll.Gal4, dpp-blink.Gal4, en.Gal4 and ptc.Gal4 (Drysdale
and Crosby, 2005). Dominant enhancement of dpps6/dpphr4 wing
phenotypes by Df(2L)C28, Mad1, Df(3R)E40 and Med7 was evaluated
by examining at least 500 individuals of each genotype. Adult
Gal4/UAS genotypes were generated using males from two
independent lines homozygous for a UAS construct crossed to Gal4
bearing females at 25°C. Tables containing quantitative data for all
Gal4/UAS genotypes are available upon request. For Gal4/UAS
combinations involving SMAD2 alleles, wing size was calculated as
described (Marquez et al., 2001). Clones of cells homozygous for the
genetic null zw3sggD127 or the protein null zw3M11 were generated by
standard methods (Siegfried et al., 1992).
Embryos and discs
Histochemical detection of ␤-galactosidase activity in embryos was
conducted as described (Newfeld et al., 1996). Embryonic cuticles
were prepared by standard methods. Vein primordia in pupal wing
imaginal discs were detected with a monoclonal antibody recognizing
Drosophila SRF (Marenda et al., 2004) or a riboprobe transcribed
from a rhomboid cDNA (Wolff, 2000). Anterior margin bristle
primordia in third instar larval wing discs were detected with a
monoclonal antibody recognizing Achaete (Skeath and Carroll, 1991).
Results
Mad1 and Med7 are gain-of-function alleles with
dominant-negative activity
The Mad1 allele intrigued us for two reasons. First, it was the
only Mad allele isolated from two screens (75,000
chromosomes in total) for modifiers of dpp adult viable
phenotypes (Sekelsky et al., 1995; Su et al., 2001). By contrast,
four alleles of Mad, including the Df(2L)C28 deletion, were
identified in a pilot screen (1850 chromosomes) for dominant
maternal enhancement of dpp embryonic lethal phenotypes
(Raftery et al., 1995). Second, in an initial genetic analysis,
Mad1 was a stronger enhancer of two dpp recessive embryonic
lethal mutations than was Df(2L)C28 (Sekelsky et al., 1995).
In re-examining the initial data on Mad1, it became clear that
certain classes of gain-of-function mutation (e.g. dominant
negative) could not be detected in an assay for enhancement of
lethality. The original data showed that heterozygosity for an
allele with a complete loss of Mad function, such as
Df(2L)C28, resulted in essentially maximum enhancement.
Only 0.5-3.0% of the expected progeny survive (Sekelsky et
al., 1995). In this assay, Mad mutant alleles with dominantnegative effects would appear to be loss-of-function alleles, as
less than 0% of the expected progeny is not possible – 0% was
seen for Mad1 enhancement of dpphr56.
We conducted a less restrictive test of the relationship
between Mad1 and dpp to determine whether Mad1 was a
dominant-negative allele. For the test, we exploited a dpp adultviable mutant phenotype (Nicholls and Gelbart, 1998). The test
is based upon a transheterozygous dpp mutant genotype that is
Development and disease
SMAD gain-of-function mutations 4885
Development
Fig. 1. Mad1 and Med7 are dominant-negative
alleles. (A) dpps6/dpphr4 wing with normal
veins. Longitudinal veins 1-5, the anterior
crossvein (acv) and the posterior crossvein
(pcv) are shown. (B) dpps6/dpphr4 Df(2L)C28
with truncated L4/L5. (C) dpps6/dpphr4 Mad1
with abnormal L2/L3/L4, no crossveins and
two margin notches. (D) dpps6/dpphr4 Med7
with a truncated L5 and two margin notches.
adult viable with occasional wing vein defects (Fig. 1A).
Truncation of Longitudinal vein 5 (L5) is observed in 11% of
flies bearing the dpps6/dpphr4 genotype, while 2% display
truncations of L4 and L5.
When we placed a single copy of Df(2L)C28 into
dpps6/dpphr4 individuals, the frequency of L5 defects increased
to 95%, 20% had defects in L4 and L5 (Fig. 1B), and 5% had
defects in L5 and L2, or L5 and the posterior crossvein. When
we placed the Mad1 allele into dpps6/dpphr4 individuals, the
frequency and severity of vein defects increased beyond that
seen with Df(2L)C28. In flies carrying Mad1, the frequency of
wings with defects in L4 and L5 increases to virtually 100%.
Furthermore, 10% of wings with L4 and L5 defects have
missing crossveins (anterior, posterior or both) and small
margin notches (Fig. 1C). We also noted that a statistically
significant amount of lethality was associated with the
dpps6/dpphr4 Mad1 genotype. Only 71% of the expected
dpps6/dpphr4 Mad1 progeny were recovered (P<0.005). No
lethality was associated with either the dpps6/dpphr4 or the
dpps6/dpphr4 Df(2L)C28 genotypes. Mad1 has a greater effect
on dpps6/dpphr4 vein phenotypes and survival than does a
deletion of Mad, indicating that Mad1 is a gain-of-function
allele with dominant-negative activity.
We then identified the lesion in Mad1 and in six additional
Mad alleles (Table 1). The Mad1 mutation is an amino acid
substitution (Q90L) due to an A to T transversion at nucleotide
615 of the cDNA. Of thirteen sequenced Mad mutant alleles
(Sekelsky et al., 1995; Chen et al., 1998) (and this report),
Mad1 is the only mutation in the MH1 domain. Given that
Mad1 is a dominant-negative allele with a lesion in the
transcriptional activation domain, we generated the following
two-part hypothesis for Mad1 dominant-negative activity. First,
we propose that the MAD1 protein is unable to activate
transcription, but is capable of receptor phosphorylation and of
forming complexes with its partner SMAD protein Medea
(MED). Second, as a result, MAD1 dominant-negative activity
derives from the formation of non-functional complexes that
deplete the pool of MED to the point that wild-type MAD
proteins are unable to find sufficient partners for normal
activity.
Circumstantial evidence for the first part of this hypothesis
is provided by two sources. First, biochemical studies of
human SMAD4 show that MH1 mutations prevent DNA
binding and transcriptional activation (Dai et al., 1998; Xu and
Attisano, 2000). Second, an examination of aligned SMAD
MH1 sequences (Newfeld et al., 1999) revealed that the amino
acid affected in the MAD1 protein, Q90, is conserved in all fly
and vertebrate SMADs that participate in DNA binding
(receptor-associated SMADs and co-SMADs), but is not
conserved in any antagonist SMADs. Furthermore, in the
structure of the SMAD3 MH1 domain bound to DNA, Q90 is
one of three residues that make direct base contact (Shi et al.,
1998). Nematode SMAD sequences show the same
relationship between Q90 and DNA binding. Q90 is present in
the unusual antagonist DAF-3. DAF-3 antagonizes TGF␤
signal transduction by binding to DNA and repressing gene
expression (Thatcher et al., 1999), a mechanism not used by
other antagonist SMADs. In addition, Q90 is not present in the
atypical receptor-associated SMADs DAF-8 and DAF-14.
Both DAF-8 and DAF-14 stimulate the expression of TGF␤
target genes by inhibiting DAF-3 function (Inoue and Thomas,
2000). Drosophila SMAD2 is the single exception. The amino
acid corresponding to Q90 in Drosophila SMAD2 falls in a run
of nine amino acids unlike any other SMAD protein.
Table 1. New Mad mutant sequences
Allele
Mad1
Mad5
Mad6
MadB65.3
Mad8
Mad11
Mad7
Lesion
Codon
Domain
Alteration
Missense
Missense
Missense
Missense
Missense
Missense
Missense
90
272
272
272
358
410
419
MH1
MH2
MH2
MH2
MH2
MH2
MH2
Gln to Leu
Arg to His
Arg to His
Arg to His
Ser to Leu
Gly to Asp
Val to Met
*Previously reported (Sekelsky et al., 1995), except for Mad1 and MadB65.3.
Effect*
Dominant negative
Loss of function (hypomorph)
Loss of function (hypomorph)
Loss of function (hypomorph)
Loss of function (hypomorph)
Loss of function (hypomorph)
Loss of function (null)
Development
4886 Development 132 (21)
Research article
In order to experimentally test our
hypothesis that the MAD1 protein is
transcriptionally inactive, we generated
UAS.Mad1 and UAS.Mad12. The Mad12
allele has a nonsense mutation (Q417Stop)
in the MH2 domain that removes the three
serines phosphorylated in response to DPP
signaling. The Mad12 allele behaves exactly
like a deletion of Mad in both genetic and
biochemical assays (Sekelsky et al., 1995;
Hoodless et al., 1996). We used UAS.Mad12
as a control for overexpression of a loss-offunction mutation. We also examined
UAS.Mad, as a control for overexpression
of the wild-type protein.
In an initial characterization, we
expressed these transgenes in wild-type
wing discs and assayed their effect on vein
formation in adult wings. We have shown,
using Mad12 mutant clones, that vein
formation is dependent on Mad activity
(Marquez et al., 2001). Expression of
UAS.Mad (Fig. 2B) (Marquez et al., 2001)
produces excess vein tissue. Alternatively,
Mad genomic loss-of-function genotypes
Fig. 2. Expression of MAD1 and SMAD4130S generates phenotypes that resemble Mad
loss-of-function phenotypes. (A) Wild-type wing. (B) UAS.Mad/ptc.Gal4 with extra vein
(transheterozygous
combinations
of
tissue between L3/L4/L5. (C) UAS.Mad12/69B.Gal4 appears wild type. (D) Mad11/Mad12
hypomorphic alleles) show reduced
with
no L2, abnormal L4/L5, no crossveins and a large margin notch (not to scale).
venation and growth defects (Fig. 2D)
1
(E)
UAS.Mad
/69B.Gal4 with truncated L2/L3/L4/L5, abnormal crossveins and a
(Sekelsky et al., 1995). Expression of the
moderate margin notch. (F) UAS.SMAD4130S/A9.Gal4 with truncated L2/L3/L4/L5 and a
12
loss-of-function allele UAS.Mad
with
small margin notch.
69B.Gal4 had no effect on any aspect of
wing development (Fig. 2C). Expression of
UAS.Mad1 with 69B.Gal4 led to vein
truncations and incomplete wing outgrowth (Fig. 2E).
In UAS.Mad/ptc.Gal4 discs, Drosophila SRF expression is
Expression of UAS.Mad with 69B.Gal4 did not lead to defects
reduced in the primordia of L3, L4 and L5 (Fig. 3B). This is
in vein formation or wing outgrowth (data not shown). The
consistent with the excess vein phenotype of
UAS.Mad1 phenotype resembles the Mad loss-of-function
UAS.Mad/ptc.Gal4 wings (Fig. 2B). In UAS.Mad12/69B.Gal4
phenotype (compare Fig. 2D with 2E).
discs, Drosophila SRF expression is essentially unaffected
In addition to effects on venation, many UAS.Mad1
(Fig. 3C). This is consistent with the wild-type appearance of
genotypes showed significant reductions in viability. For
UAS.Mad12/69B.Gal4 wings (Fig. 2C). In UAS.Mad1/69B.Gal4
1
example, 52% of the expected UAS.Mad /T80.Gal4 flies, 55%
discs, Drosophila SRF expression is expanded in the primordia
of the UAS.Mad1/dll.Gal4 flies and 69% of the
of L3, L4 and L5 (Fig. 3D). This is consistent with the reduced
UAS.Mad1/24B.Gal4 flies were observed (P<0.005).
venation of UAS.Mad1/69B.Gal4 wings (Fig. 2E). These results
12
Expression of UAS.Mad or UAS.Mad had no effect on
suggest that MAD1 is incapable of activating rho and,
viability. These studies are consistent with our genetic analysis
furthermore, that MAD1 protein can block the ability of
of Mad1, supporting its identification as a dominant-negative
endogenous MAD to activate rho.
allele.
Examination of rho transcript accumulation in pupal wing
To further test our hypothesis that the MAD1 protein is
discs expressing UAS.Mad1 confirms this interpretation. In
transcriptionally inactive, we examined two molecular markers
UAS.Mad1 discs, rho expression is reduced in the primordia of
for vein formation in wing imaginal discs. We analyzed
L3, L4 and L5. L4 expression is the most severely affected
Drosophila Serum response factor (SRF; Blistered – FlyBase)
(Fig. 3F). This is consistent with the vein phenotype of
protein expression and rhomboid (rho) transcript
UAS.Mad1/69B.Gal4 wings (Fig. 2E). Because rho may be a
accumulation. Drosophila SRF expression is widespread in
direct transcriptional target of MAD, this result supports our
third instar wing discs but its expression is downregulated in
hypothesis that the MAD1 protein cannot activate transcription.
vein primordia by rho in pupal wing discs (Fig. 3A) (Biehs et
We then noted that of the 18 sequenced Med mutant alleles
al., 1998). rho transcription in vein primordia may be directly
only Med7 is an MH1 domain mutation (Das et al., 1998;
dependent upon DPP signaling via MAD (Fig. 3E) (Yu et al.,
Hudson et al., 1998; Wisotzkey et al., 1998). Furthermore, the
1996). Thus, in vein development, the expansion of Drosophila
C99S mutation in Med7 alters one of three cysteine residues
SRF expression indicates a lack of rho activity and the absence
that is predicted to coordinate a zinc atom (Chai et al., 2003).
of rho activity indicates that it is not being transcribed by
We tested Med7 and the Df(3R)E40 deletion of Med in our
MAD.
assay for enhancement of the dpps6/dpphr4 wing phenotype. In
Development and disease
SMAD gain-of-function mutations 4887
Development
Just two out of 33 sequenced mutations (6%) occur
in the MH1 even though this domain covers roughly
23% of the amino acids in these proteins.
SMAD4130S and SMAD4100T are gain-offunction alleles associated with human
tumors
Our identification of Mad1 and Med7 as gain-offunction alleles with MH1 mutations suggested that
SMAD MH1 mutations identified in human tumors
might also generate gain-of-function alleles. We
tested this hypothesis with a set of five SMAD
mutant cDNAs derived from pancreas and colon
tumors (Table 2).
In studies with transgenes expressing wild-type
SMAD2 or SMAD4, we showed that their
phenotypes mirrored those of their fly homologs,
Drosophila SMAD2 and MED, respectively
(Marquez et al., 2001). This suggested to us that
human SMAD proteins expressed in flies function in
the same manner as their fly homologs do. By
extending this idea to SMAD tumor alleles, we
predict that the manner in which a mutation affects
the activity of a human SMAD protein expressed in
flies will inform us about the effect of that mutation
on the human SMAD protein.
When SMAD alleles with mutations in the MH2
domain (UAS.SMAD4524ST, UAS.SMAD2⌬344-358 and
UAS.SMAD4493H) are expressed in flies, their wings
Fig. 3. Expression of MAD1 results in expanded Drosophila SRF expression as
are wild type in size and appearance like those
a result of reduced rhomboid transcription. (A) Wild-type pupal wing disc
stained for Drosophila SRF expression. Anterior is towards the top and distal
expressing the loss-of-function allele UAS.Mad12.
to the left. L1-L5 primordia are indicated. (B) UAS.Mad/ptc.Gal4 has
Alternatively, wings expressing the MH1 missense
expanded regions without Drosophila SRF corresponding to L3/L4.
mutation UAS.SMAD4130S (Fig. 2F) are similar to
(C) UAS.Mad12/69B.Gal4 appears wild type. (D) UAS.Mad1/69B.Gal4 with
those expressing the dominant-negative allele
expanded regions of Drosophila SRF that limit the extent of L3/L4. (E) WildUAS.Mad1 (Fig. 2E). Both UAS.SMAD4130S and
type pupal wing disc stained for rhomboid (rho) transcripts. L3/L4/L5
UAS.Mad1 engender defects in vein formation and
primordia are indicated. rho expression in L1/L2 is not yet fully developed at
wing outgrowth. Furthermore, UAS.SMAD4130S
this stage; note the difference in the visibility of L1/L2 versus L3/L4/L5 in A.
1
expression results in reduced viability like UAS.Mad1
(F) UAS.Mad /69B.Gal4 with reduced rho expression. Expression in L4 is
does. For example, UAS.SMAD4130S/dll.Gal4 flies
severely reduced and distal truncations in L3/L5 are visible.
were recovered at 36% and UAS.Mad1/dll.Gal4 flies
were recovered at 55% of the expected frequency.
this assay, Df(3R)E40 increased the frequency of dpps6/dpphr4
Results for SMAD4130S suggest that it too is a gain-of-function
wings with L5 truncations from 11% to 28%. When we placed
allele with dominant-negative activity.
the Med7 allele into dpps6/dpphr4 individuals, the frequency and
The expression of the MH1 missense allele SMAD4100T
severity of vein defects increased beyond that seen with
generated a wing phenotype never before reported in any study
Df(3R)E40. In flies carrying Med7, the frequency of wings with
of TGF␤ signaling in flies. Most wings expressing
defects in L5 and L4 was 42%. Furthermore, 15% of these
UAS.SMAD4100T strongly throughout the wing blade (e.g.
wings also have defects in L2, or the posterior crossvein or
MS1096.Gal4, C765.Gal4 or T80.Gal4) have ectopic
margin notches (Fig. 1D). Med7 is also a gain-of-function allele
mechanosensory bristles on the blade (Fig. 4A). In wild type,
with dominant-negative activity.
two rows of mechanosensory bristles (stout and thin) are
Perhaps dominant-negative activity explains the relative
normally found in the triple row region of the proximal anterior
infrequency of detectable MH1 mutations in Mad and Med.
wing margin (Couso et al., 1994). The ectopic mechanosensory
Table 2. Human SMAD tumor alleles expressed in flies
Allele
SMAD4100T
SMAD4130S
SMAD4493H
SMAD4524ST
SMAD2⌬344-358
Lesion
Codon
Domain
Alteration
Tumor
Reference
Missense
Missense
Missense
Nonsense
Deletion
100
130
493
524
344-358
MH1
MH1
MH2
MH2
MH2
Arg to Thr
Pro to Ser
Asp to His
Cys to Stop
14aa deletion
Pancreas
Colorectal
Pancreas
Colorectal
Colorectal
Schutte et al., 1996
Thiagalingam et al., 1996
Hahn et al., 1996
Riggins et al., 1996
Riggins et al., 1996
4888 Development 132 (21)
Research article
Development
Fig. 4. Expression of SMAD4100T induces ectopic mechanosensory bristles on the
wing blade. (A) UAS.SMAD4100T/MS1096.Gal4 wing with six ectopic mechanosensory
bristles (open arrowheads). This image is a composite of three wings such that all
positions for ectopic bristles are shown (the three bristles on medial L3 never appear
together). (B) Dorsal L1 of wild type with two campaniform sensilla (arrowheads).
(C) Dorsal L1 of UAS.SMAD4100T/MS1096.Gal4 with a stout mechanosensory bristle
replacing Twin Sensillum One (open arrowhead; black arrowhead indicates Twin
Sensillum Two). (D) Ventral L3 of wild type with a campaniform sensillum
(arrowhead). (E) Ventral L3 of
UAS.SMAD4100T/MS1096.Gal4 with a
stout mechanosensory bristle
replacing the sensillum (open
arrowhead). (F) Dorsal L3 of wild
type with three campaniform sensilla
(arrowheads). (G) Dorsal L3 of
UAS.SMAD4100T/MS1096.Gal4 with a
thin mechanosensory bristle replacing
the proximal sensillum (open
arrowhead). (H) Dorsal L3 of
UAS.SMAD4100T/MS1096.Gal4 with a
thin mechanosensory bristle replacing
the middle sensillum (open
arrowhead). (I) Ectopic thin
mechanosensory bristles are seen on
a wing with small, unmarked clones
of cells homozygous for zw3M11
(open arrowheads).
bristles seen in wings expressing UAS.SMAD4100T are largely
derived from the transformation of normally ‘bristle-less’
mechanosensory receptors called campaniform sensilla (Held,
2002).
Two campaniform sensilla (Twin Sensilla One and Two) are
located on the dorsal surface of L1 (Fig. 4B). The most
frequent
transformation
seen
in
UAS.SMAD4100T/
MS1096.Gal4 wings is a stout mechanosensory bristle in place
of Twin Sensillum One (Fig. 4C). A sensillum on the ventral
surface of L3 has only recently been reported (Aigouy et al.,
2004) (Fig. 4D). A stout mechanosensory bristle is
occasionally seen in the place of this sensillum on a
UAS.SMAD4100T/MS1096.Gal4 wing (Fig. 4E). Three sensilla
(Sensilla Campaniformium of Dorsal Radius) are located on
the dorsal surface of L3 (Fig. 4F). The second most frequent
transformation seen in UAS.SMAD4100T/MS1096.Gal4 wings is
a thin mechanosensory bristle replacing either the proximal
(Fig. 4G), middle (Fig. 4H) or distal (not shown) sensillum at
roughly equal frequencies. The most posterior ectopic bristle
on a UAS.SMAD4100T/MS1096.Gal4 wing is rare, and is located
on the ventral surface of L5 (Fig. 4A). We could not detect a
campaniform sensillum in this location and none has been
reported. Thus, five out of the six ectopic bristles on
UAS.SMAD4100T/MS1096.Gal4 wings are derived from the
transformation of sensilla.
Though never previously associated with TGF␤ signaling,
the presence of ectopic anterior margin bristles on the wing
blade is not unprecedented. Wingless (WG) is a secreted
signaling molecule expressed along the presumptive wing
margin in imaginal discs. Numerous studies have shown that
mechanosensory bristle development along the margin requires
WG as well as components of the canonical WG signaling
pathway (Couso et al., 1994). Alternatively, when ectopic WG
signaling is activated in the presumptive wing blade, for
example in zeste white 3 (zw3; shaggy, sgg – FlyBase) null
clones, ectopic mechanosensory bristles result (Blair, 1992).
Several thin mechanosensory bristles on the wing blade
emanating from unmarked clones of zw3M11 cells are shown
(Fig. 4I). Ectopic bristles can be generated anywhere on the
wing blade by zw3M11 clones.
The unexpected transformation of sensilla to bristles on
wings expressing UAS.SMAD4100T suggests the hypothesis that
the R100T mutation in SMAD4100T conveys a novel activity
upon the encoded protein. Thus, SMAD4100T is the second gainof-function allele associated with a human tumor. Furthermore,
the similarity between the UAS.SMAD4100T wing phenotype
and that of zw3M11 clones suggests a second hypothesis – that
SMAD4100T has the ability to activate WG target genes.
To test our hypothesis that the R100T mutation conveys a
novel activity upon SMAD4, we examined wings expressing
Drosophila TGF␤ family members with known roles in wing
development (DPP and GBB) (Ray and Wharton, 2001). We
wondered whether these genes could generate ectopic bristles
on the wing blade. In these experiments, UAS.Dpp expression
was absolutely lethal, and significant lethality was also
associated with UAS.Gbb expression. In the two experiments
where adult flies were obtained, we found that UAS.Gbb
inhibits triple row bristle formation. ap.Gal4 is expressed in all
dorsal cells of the wing disc (Diaz-Benjumea and Cohen,
1993). We observed that the proximal 25% of the anterior
margin of a UAS.Gbb/ap.Gal4 wing has no bristles (Fig. 5A).
More distally, the triple row reappears but is quite irregular
(Fig. 5B,C). When UAS.Gbb was driven in all cells of the wing
blade with C765.Gal4 (de Celis et al., 1996), the phenotype
was similar but less severe (data not shown). In summary, none
of our studies of UAS.Dpp or UAS.Gbb generated sensilla to
Development and disease
SMAD gain-of-function mutations 4889
Development
Fig. 5. Expression of GBB does not induce ectopic anterior margin bristles on the wing blade. (A) Ventral view of a UAS.Gbb/ap.Gal4 wing
with all bristles missing from the proximal anterior wing margin (arrowhead). A distal portion of the anterior wing margin, indicated by the
black bar, is shown at higher magnification in B,C. (B) Dorsal view: the row of stout mechanosensory bristles appears in a roughly wild-type
pattern with occasional gaps. The row of widely spaced chemosensory bristles shows considerable irregularity in spacing, with some bristles
displaced dorsally. (C) Ventral view: the row of alternating chemosensory and thin mechanosensory bristles is disorganized and numerous thin
mechanosensory bristles are present in a region ventral to their normal location.
bristle transformation, supporting our hypothesis that
UAS.SMAD4100T is a gain-of-function allele with novel activity.
To test the second hypothesis (that the novel activity of the
SMAD4100T protein is the ability to activate WG target genes),
we conducted three experiments. In the first, we tested the
ability of SMAD4100T to suppress phenotypes generated by
DN-TCF, a dominant-negative form of the WG pathway
transcription factor Drosophila TCF (Pangolin, PAN –
FlyBase). For this test, we used MS1096.Gal4, a driver strongly
expressed throughout the wing blade. The wings of UAS.DNTCF/MS1096.Gal4 flies (Fig. 6A) are very similar to those
with impaired WG signaling (e.g. Couso et al., 1994). These
wings are very small and lack a margin. However, if two (Fig.
6B) or three (Fig. 6C) copies of UAS.SMAD4100T are also
present, then the UAS.DN-TCF/MS1096.Gal4 wing phenotype
is visibly suppressed in a dosage-dependent fashion. If instead
we add copies of UAS.lacZ, then the UAS.DNTCF/MS1096.Gal4 wing phenotype is unaffected (data not
shown). This result suggests that, even in the presence of a
strong antagonist of WG signaling, SMAD4100T is capable of
activating WG target genes involved in wing growth and
margin formation.
We made two additional observations in these experiments.
First, siblings to our experimental flies (those without
UAS.DN-TCF) showed dosage-dependant dominant-negative
phenotypes for DPP-dependent processes such as vein
formation (Fig. 6D-F). Thus, above a certain threshold,
UAS.SMAD4100T resembles the other MH1 mutations in our
study – it has dominant-negative effects on DPP signaling.
Second, we found that the UAS.DN-TCF/MS1096.Gal4
Fig. 6. Expression of SMAD4100T
suppresses wing phenotypes generated
by dominant-negative Drosophila
TCF. (A) UAS.DN-TCF/MS1096.Gal4
wing is only 10% of the size of a wildtype wing and has no margin.
(B) UAS.DN-TCF/MS1096.Gal4 wing
with two copies of UAS.SMAD4100T is
roughly three-fold larger than the wing
in A, and the anterior margin is
restored. Transformation of Twin
Sensillum One into a bristle is noted
(arrowhead). (C) UAS.DNTCF/MS1096.Gal4 wing with three
copies of UAS.SMAD4100T is roughly
four-fold larger than the wing in A,
and the anterior margin is restored.
Sensillum-to-bristle transformation is
noted (arrowhead).
(D) UAS.SMAD4100T/MS1096.Gal4
wing has a normal margin and two
sensilla-to-bristle transformations
(arrowheads). (E) Wing with
MS1096.Gal4 driving two copies of
UAS.SMAD4100T has a normal margin, sensilla-to-bristle transformation (arrowhead) and vein defects. (F) Wing with MS1096.Gal4 driving
three copies of UAS.SMAD4100T has a reduced size, a normal margin, sensilla-to-bristle transformation (arrowhead) and vein defects.
Development
4890 Development 132 (21)
Research article
genotype is male-specific lethal. In a cross of MS1096.Gal4
homozygous females to UAS.DN-TCF homozygous males, we
obtained 199 female progeny and no male progeny. By using
the F1 females in crosses to males containing various numbers
of UAS.SMAD4100T insertions, we obtained male progeny
bearing MS1096.Gal4 and UAS.DN-TCF. The suppression of
male-specific lethality suggests that SMAD4100T is able to
activate WG target genes outside of wing discs.
To further support this observation, we expressed
SMAD4100T in the embryonic ventral epidermis. In most
abdominal segments, the ventral epidermis is composed of
twelve rows of cells: six rows that secrete smooth cuticle
followed by six rows that secrete protrusions called denticles
(Fig. 7A). Cells choose to secrete naked cuticle or denticles
according to positional information supplied, in part, by WG
and Engrailed (EN) (Gritzan et al., 1999; Alexandre et al.,
1999). WG signals instruct cells to secrete naked cuticle. EN
is expressed just posterior to WG and an EN-expressing cell
secretes the first denticle row (Fig. 7B). When a constitutively
active form of the WG pathway signal transducer Armadillo
(CA-ARM) is expressed via en.Gal4, the first row of denticles
in all segments is transformed into naked cuticle (Fig. 7C).
When SMAD4100T is expressed with en.Gal4, patches of cells
within the first denticle row are transformed into naked cuticle
on one or more segments of most embryos (Fig. 7D). This
result is consistent with a recent report that weak global
expression of CA-ARM results in a patchy loss of denticles
(Hayward et al., 2005). We conclude that SMAD4100T is
capable of phenocopying activated WG signaling in the ventral
epidermis. These data support the idea that the ability of
SMAD4100T to activate WG target genes is not context
dependent.
Moving from the phenotypic to the molecular level, we
Fig. 7. Expression of SMAD4100T mimics the expression of constitutively active
ARM. (A) Wild-type six-row denticle pattern shown with anterior to the left. The
small, anteriorly pointed denticles of row one are indicated (arrowhead).
(B) UAS.lacZ/en.Gal4 six-row denticle pattern; lacZ expression is coincident with
row one (arrowhead). (C) UAS.CA-ARM/en.Gal4 five-row denticle pattern. The
transformation of row one denticles into naked cuticle is indicated (arrowhead).
(D) UAS.SMAD4100T/en.Gal4 six-row denticle pattern. The transformation of a
subset of row one denticles into naked cuticle is indicated (arrowhead).
Fig. 8. Expression of SMAD4100T activates the
expression of the WG target gene achaete. (A) Wildtype third instar wing disc stained for Achaete (AC).
a, anterior; m, proximal margin; v, ventral surface;
dis, distal margin. (B) zw3M11 clones produce ectopic
AC expression on the presumptive blade (arrowheads).
(C) UAS.SMAD4100T/C765.Gal4 with ectopic AC
expression on the presumptive blade (arrowheads).
(D) UAS.Dpp/C765.Gal4. This is a lethal genotype. The
disc is overgrown (image shown at a reduced
magnification) but it has wild-type AC expression.
(E) UAS.Gbb/C765.Gal4 with reduced AC expression in
the proximal anterior margin primordia (arrowhead).
Development and disease
looked directly at the expression of Achaete (AC) in wing discs
expressing SMAD4100T. AC is expressed in the presumptive
anterior wing margin (Fig. 8A) coincident with WG. Genetic
analyses have shown that ac is required for the development of
the margin bristles and that ac is a direct target of WG signaling
(Skeath and Carroll, 1991; Blair, 1992). As a result, wing discs
bearing clones of cells homozygous for a mutation in zw3 have
numerous regions of ectopic AC expression in the presumptive
wing blade (Fig. 8B). The same result is seen when examining
AC expression in wing discs from individuals expressing
UAS.SMAD4100T (Fig. 8C). In a UAS.DPP-expressing disc,
the pattern of AC expression along the presumptive margin is
normal, although the disc is overgrown, reflecting the ability
of DPP to influence wing growth (Fig. 8D). In a UAS.GBBexpressing disc, AC expression is absent in the most proximal
region of the anterior margin, reflecting the ability of GBB to
inhibit bristle formation in this region (Fig. 8E). Overall, our
studies of DN-TCF, CA-ARM and AC strongly support the
hypothesis that the SMAD4100T protein is capable of activating
the expression of WG pathway target genes.
Development
Discussion
From a larger perspective, our study establishes guidelines for
interpreting data from transgenic analyses of human tumor
alleles. In principle, studies of this type can be applied to
mutant alleles of any well-conserved tumor suppressor gene or
oncogene. Furthermore, our unexpected finding that all of the
tested mutations in the DNA-binding domain of SMAD genes
are gain of function, whereas all of the tested mutations in the
multimerization domain are loss of function, indicates that
missense mutations in modular proteins should be
experimentally characterized rather than defaulted to the lossof-function category.
For SMAD tumor suppressor genes, our identification of two
gain-of-function alleles of SMAD4 (dominant negative and
neomorphic) falsifies the prevailing hypothesis that all SMAD
tumor mutations are loss-of-function mutations. Instead, our
data support an alternative hypothesis: that there are multiple
classes of SMAD mutation and that each class is associated
with a different mechanism of tumorigenesis.
This alternative hypothesis may also apply to other TGF␤
signaling pathway components with tumor-associated
mutations. For example, mutations in TGF␤ receptors are
found in tumors from the same tissues that exhibit SMAD
mutations [e.g. pancreas (Hempen et al., 2003), colon
(Peltomaki, 2001), breast (Pouliot and LaBrie, 1999), lung
(Zhang et al., 2004)]. However, missense mutations in TGF␤
receptors conferring gain-of-function activity have not been
identified in tumors, because the most common mutational
assays are loss of expression and polyA tract sequencing.
An oncogenic mechanism of tumorigenesis for
SMAD4100T
Our data for SMAD4100T are unprecedented in studies of TGF␤
signaling in flies. This suggests that SMAD4100T may induce
tumors in humans by an unexpected method. The fact that
SMAD4100T expression mimics activated WG signaling and
suppresses an antagonist of WG signaling further suggests that
SMAD4100T utilizes a mechanism of tumorigenesis associated
with loss-of-function mutations in Adenomatous Polyposis
SMAD gain-of-function mutations 4891
Coli (APC). A model based on this interpretation is shown in
Fig. 9.
In vertebrates and flies, APC serves as a component of the
highly conserved WG/int-1 (WNT) signal transduction
pathway. Like ZW3 and its homolog Glycogen Synthase
Kinase-3␤ (GSK3␤), APC functions as a WNT antagonist via
participation in a cytoplasmic retention complex that prevents
Armadillo (or its vertebrate homolog ␤-catenin) from
accumulating in the nucleus in the absence of WNT signals.
Studies in flies have shown that homozygous null clones
bearing mutations in any member of the retention complex
(ZW3, Drosophila APC and Drosophila Axin) lead to the same
phenotype: ectopic anterior margin bristles on the wing blade
as a result of the unregulated expression of WG target genes
such as AC (Blair, 1992; Akong et al., 2002; Hamada et al.,
1999). First identified in the rare inherited cancer Familial
Adenomatous Polyposis, homozygous mutations in APC are
now found in roughly 85% of all colon tumors (Kinzler and
Vogelstein, 1996). In studies of mice engineered to
homozygose APC null mutations only in cells of their intestinal
epithelium, the immediate consequence of APC loss was
ectopic expression of WNT target genes via constitutively
nuclear ␤-catenin (Sansom et al., 2004).
Given their roles in their respective signal transduction
pathways, loss-of-function mutations in APC cause tumors by
a fundamentally different mechanism than loss-of-function
Fig. 9. SMAD4100T may cause tumors via an ‘APC-like’ mechanism
not previously associated with defective TGF␤ signaling. Model for
the mechanism of tumorigenesis used by SMAD4100T based upon its
ability to activate WG target genes in flies. (Left) In the model,
SMAD4100T functions in a way that mimics the repression of the
ARM-destruction complex (ZW3, APC and Axin) in the WG
pathway. In this illustration, we show SMAD4100T actively inhibiting
the destruction complex, but SMAD4100T may interact with the WG
pathway at other points, such as target promoters. SMAD4100T and
proteins potentially affected by its activity are shown in red. (Right)
The mechanism of tumorigenesis associated with loss-of-function
mutations in APC. Loss of APC activity inhibits the ␤-catenin
destruction complex (GSK3␤, APC and Axin) leading to the
overexpression of WNT target genes and Familial Adenomatous
Polyposis. APC and proteins affected by the loss of APC function are
shown in red.
Development
4892 Development 132 (21)
SMAD mutations. Specifically, inactive APC proteins cannot
block a mitogenic WNT signal (an oncogenic mechanism),
whereas inactive SMAD proteins cannot transduce an antimitotic TGF␤ signal (a tumor suppressor mechanism).
Interestingly, one study of SMAD4100T in mammalian cells
suggested that this allele could employ an oncogenic
mechanism of tumorigenesis (Dai et al., 1999), a proposal
consistent with our data.
An examination of the primary difference between the
phenotypes of SMAD4100T and zw3, Apc and Axin mutant clones
may shed light on SMAD4100T-associated tumor formation. The
primary difference is the location of ectopic bristles on the wing
blade. In wings expressing SMAD4100T, ectopic mechanosensory
bristles are derived from a cell fate transformation of
mechanosensory receptors (campaniform sensilla). Cells extruding
sensilla and those extruding bristles are derived from Sensory
Organ Precursor cells (Aigouy et al., 2004). Alternatively, zw3, Apc
and Axin mutant clones generate cell fate transformations
anywhere on the wing blade – regardless of cell lineage. This
discrepancy suggests that SMAD4100T is not as potent an activator
of WNT target genes as zw3, Apc or Axin mutations. SMAD4100T
may only activate WNT target genes in cells predisposed to
tumorigenesis, perhaps by pre-existing mutations.
This possibility is supported by our studies of wings with an
increasing dosage of UAS.SMAD4100T. When wild-type WG
signaling is present, increasing the UAS.SMAD4100T copy
number did not increase the frequency of sensillum to bristle
transformation. However, in wings with reduced WG signaling
due to the activity of UAS.DN-TCF, increasing the copy
number of UAS.SMAD4100T quantitatively increased the
rescue of WG-dependent functions outside of sensilla (e.g. in
wing outgrowth and anterior margin formation).
Molecular nature of the SMAD4100T-WNT pathway
interaction
The
transformation
of
campaniform
sensilla
to
mechanosensory bristles was reported once previously. Several
transheterozygous mutant genotypes of ash2, a member of the
Trithorax group of transcriptional regulators, generate this
phenotype (Adamson and Shearn, 1996). Although the ASH2
protein contains a zinc finger motif, its function has not yet
been demonstrated biochemically. Studies of its yeast homolog
suggest that ASH2 may function in chromatin remodeling and
transcriptional activation as part of a complex containing
histone methyltransferases (Janody et al., 2004). The similarity
of SMAD4100T and ash2 mutant phenotypes, and the ability of
SMAD4100T to suppress DN-TCF phenotypes, suggest that
SMAD4100T contributes to the activation of WNT target genes
downstream of APC, perhaps by participating in transcription
factor complexes.
Three previous reports have shown physical interactions
between the wild-type SMAD proteins and transcriptional
effectors of WNT signaling (␤-catenin and TCF). One study
used Xenopus embryos to demonstrate that SMAD4/␤catenin/TCF complexes activate the transcription of the WNT
target gene twin (Nishita et al., 2000). Recently, SMAD1/␤catenin/TCF complexes were detected in renal medullary
dysplasia in ALK3 transgenic mice (Hu et al., 2003), and in
human dysplastic renal tissue (Hu and Rosenblum, 2004). We
are currently testing the possibility that SMAD4100T cooperates
with ARM and/or TCF to activate the transcription of AC.
Research article
Clinical implications
At this time, therapeutic research on SMAD-associated tumors
is guided by the current hypothesis that all SMAD mutations
lead to tumors via a loss of a TGF␤-encoded anti-mitogenic
signal. As a result, effort is focused on restoring the wild-type
function in tumors by gene replacement. However, we have
shown that two SMAD4 tumor alleles are gain-of-function
mutations. One important feature of gain-of-function mutations
is that they exert their effect even in the presence of a wild-type
allele on the homologous chromosome. Thus, it seems unlikely
that gene replacement will be successful in inhibiting
tumorigenesis in cells with SMAD4 gain-of-function mutations
In individuals with APC mutant colon tumors (those with
unregulated WG target gene expression such as Familial
Adenomatous Polyposis), the transition from adenomatous
polyps to carcinoma will take roughly ten years. Alternatively,
for TGF␤ receptor mutant colon tumors [those unable to
respond to a TGF␤-encoded anti-mitogenic signal such as
Hereditary Non-Polyposis Colorectal Cancer (HNPCC)],
progress from adenomatous polyps to carcinoma takes less
than three years (Souza, 2001). Given these data, if SMAD4100T
induces tumorigenesis by an ‘APC-like’ mechanism while
SMAD4 dominant-negative and loss-of-function alleles
stimulate tumorigenesis by an ‘HNPCC-like’ mechanism, then
the prognosis for cancer patients with a SMAD4100T mutation
is distinctly different from that of patients with other SMAD
mutations.
This raises several issues for future investigation. First, how
many different mutations in SMAD4 can generate an ‘APClike’ gain-of-function allele? To date, three SMAD4 missense
mutations near codon 100 have been identified in colon tumors
[Y95N, C115R and N118K (Iacobuzio-Donahue et al., 2004)].
All of these mutations (including R100T) occur in the L2/L4
double-loop region identified in the crystal structure of the
SMAD3 MH1 bound to DNA. This loop occurs at the surface
of the molecule and is important for macromolecular
interactions (Shi et al., 1998). Are these also gain-of-function
mutations? Second, what is the relative frequency of ‘APClike’ gain-of-function SMAD4 alleles versus ‘HNPCC-like’
loss-of-function alleles in tumors from various tissues? Third,
can an accurate and efficient diagnostic test be developed to
distinguish between ‘APC-like’ and ‘HNPCC-like’ alleles in
tumors with a SMAD4 mutation? Answering these questions
will require a continued collaboration between model organism
geneticists and oncologists.
We thank G. Riggins, M. Schutte and R. Ray for cDNAs, and the
Developmental Studies Hybridoma Bank for antibodies. T. Haerry, S.
Hayashi, M. Hoffmann, A. Manoukian, L. Marsh, L. Raftery, E.
Siegfried, K. Wharton and the Bloomington Stock Center contributed
flies. A. Schmid and C. Lorson provided technical advice. A. Johnson
generated the UAS.Mad1 plasmid and assisted with image analysis.
B. Johnson, M. Stinchfield, B. Celaya and N. Emmert helped with fly
pushing. This study was supported by the NIH (CA095875 to S.J.N.).
M.B.O. is an Associate Investigator of the Howard Hughes Medical
Institute.
References
Adamson, A. and Shearn, A. (1996). Molecular genetic analysis of ash2, a
member of the Trithorax group required for imaginal disc pattern formation.
Genetics 144, 621-633.
Development
Development and disease
Aigouy, B., Van de Bor, V., Boeglin, M. and Giangrande, A. (2004). Timelapse and cell ablation reveal the role of cell interactions in fly glia migration
and proliferation. Development 131, 5127-5138.
Akong, K., Grevengoed, E., Price, M., McCartney, B., Hayden, M.,
DeNofrio, J. and Peifer M. (2002). Drosophila APC2 and APC1 play
overlapping roles in Wingless signaling in the embryo and imaginal discs.
Dev. Biol. 250, 91-100.
Alexandre, C., Lecourtois, M. and Vincent, J.-P. (1999). Wingless and
Hedgehog pattern Drosophila denticle belts by regulating the production of
short-range signals. Development 126, 5689-5698.
Barnard, J., Lyons, R. and Moses, H. (1990). The cell biology of TGF-␤.
Biochem. Biophys. Acta 1032, 79-87.
Biehs, B., Sturtevant, M. and Bier, E. (1998). Boundaries in the Drosophila
wing imaginal disc organize vain-specific genetic programs. Development
125, 4245-4257.
Blair, S. (1992). Shaggy (zeste-white 3) and the formation of supernumerary
bristle precursors in the developing wing blade of Drosophila. Dev. Biol.
152, 263-278.
Brand, A. and Perrimon, N. (1993). Targeted gene expression as a means of
altering cell fates and generating dominant phenotypes. Development 118,
401-415.
Chai, J., Wu, J., Yan, N., Massagué, J., Pavletich, N. and Shi, Y. (2003).
Features of Smad3 MH1-DNA complex: role of water and zinc in DNA
binding. J. Biol. Chem. 278, 20327-20331.
Chen, Y., Riese, M., Killinger, M. and Hoffmann, F. (1998). A genetic screen
for modifiers of Drosophila dpp signaling identifies mutations in punt, Mad
and the BMP-7 homologue, 60A. Development 125, 1759-1768.
Couso, J., Bishop, S. and Martinez-Arias, A. (1994). The wingless signaling
pathway and the patterning of the wing margin in Drosophila. Development
120, 621-636.
Dai, J., Turnacioglu, K., Schutte, M., Sugar, A. and Kern, S. (1998). DPC4
transcriptional activation and dysfunction in cancer cells. Cancer Res. 58,
4592-4597.
Dai, J., Bansal, R. and Kern, S. (1999). G1 cell cycle arrest and apoptosis
induction by nuclear Smad4/DPC4: phenotypes reversed by a tumorigenic
mutation. Proc. Natl. Acad. Sci. USA 96, 1427-1432.
Das, P., Maduzia, L., Wang, H., Finelli, A., Cho, S., Smith, M. and Padgett,
R. (1998). The Drosophila gene Medea demonstrates the requirement for
different classes of Smads in Dpp signaling. Development 125, 1519-1528.
de Celis, J., Barrio, R. and Kafatos, F. (1996). A gene complex acting
downstream of dpp in Drosophila wing morphogenesis. Nature 381, 421424.
Diaz-Benjumea, F. and Cohen, S. (1993). Interaction between dorsal and
ventral cells in the imaginal disc directs wing development in Drosophila.
Cell 75, 741-752.
Drysdale, R. A. and Crosby, M. A. (2005). FlyBase: genes and gene models.
Nucl. Acids Res. 33, D390-D395.
Eppert, K., Scherer, S., Ozcelik, S., Pirone, R., Hoodless, P., Kim, H., Tsui,
L., Bapat, B., Gallinger, S., Andrulis, I. et al. (1996). MADR2 maps to
18q21 and encodes a TGF-␤ regulated MAD-related protein that is
functionally mutated in colorectal carcinoma. Cell 86, 543-552.
Fynan, T. and Reiss. M. (1993). Resistance to inhibition of cell growth by
TGF-␤ and its role in oncogenesis. Crit. Rev. Oncog. 4, 493-540.
Gritzan, U., Hatini, V. and DiNardo, S. (1999). Mutual antagonism between
signals secreted by adjacent Wg and En cells leads to specification of
complementary regions of the Drosophila parasegment. Development 126,
4107-4115.
Hahn, S., Schutte, M., Hoque, A., Moskaluk, C., Da Costa, L., Rozenblum,
E., Weinstein, C., Fischer, A., Yeo, C., Hruban, R. et al. (1996). DPC-4:
a candidate tumor suppressor gene at human chromosome 18q21. Science
271, 350-353.
Hamada, F., Tomoyasu, Y., Takatsu, Y., Nakamura, M., Nagai, S., Suzuki,
A., Fujita, F., Shibuya, H., Toyoshima, K., Ueno, N. et al. (1999).
Negative regulation of Wg signaling by Daxin, Drosophila homolog of axin.
Science 283, 1739-1742.
Hayward, P., Brennan, K., Sanders, P., Balayo, T., DasGupta, R.,
Perrimon, N. and Martinez-Arias, A. (2005). Notch modulates Wnt
signaling by associating with Armadillo/ß-catenin and regulating its
transcriptional activity. Development 132, 1819-1830.
Held, L. (2002). Imaginal Discs: The Genetic and Cellular Logic of Pattern
Formation. Cambridge: Cambridge University Press.
Hempen, P., Zhang, L., Bansal, R., Iacobuzio-Donahue, C., Murphy, K.,
Maitra, A., Vogelstein, B., Whitehead, R., Markowitz, S., Willson, J. et
al. (2003). Evidence of selection for clones having genetic inactivation of
SMAD gain-of-function mutations 4893
the activin A type II receptor gene in gastrointestinal cancers. Cancer Res.
63, 994-999.
Hoodless, P., Haerry, T., Abdollah, S., Stapleton, M., O’Connor, M.,
Attisano, L. and Wrana, J. (1996). MADR1, a MAD-related protein that
functions in BMP2 signaling pathways. Cell 85, 489-500.
Howe, J., Roth, S., Ringold, J., Summers, R., Jarvinen, H., Sistonen, P.,
Tomlinson, I., Houlston, R., Bevan, S., Mitros, F. et al. (1998).
Mutations in the Smad4/DPC4 gene in juvenile polyposis. Science 280,
1086-1088.
Hu, M. and Rosenblum, N. (2004). Smad1, ␤-catenin and Tcf4 associate in
a molecular complex with the myc promoter in dysplastic renal tissue and
cooperate to control myc transcription. Development 132, 215-225.
Hu, M., Piscione, T. and Rosenblum, N. (2003). Elevated Smad1/␤-catenin
complexes and renal medullary dysplasia in ALK3 transgenic mice.
Development 130, 2753-2766.
Hudson, J., Podos, S., Keith, K., Simpson, S. and Ferguson, E. (1998). The
Drosophila Medea gene is required downstream of Dpp and encodes a
functional homolog of human Smad4. Development 125, 1407-1420.
Iacobuzio-Donahue, C., Song, J., Parmiagiani, G., Yeo, C., Hruban, R. and
Kern, S. (2004). Missense mutations of MADH4: characterization of the
mutational hot spot and functional consequences in human tumors. Clin.
Cancer Res. 10, 1597-1604.
Inoue, T. and Thomas, J. (2000). Targets of TGF-␤ signaling in C. elegans
dauer formation. Dev. Biol. 217, 192-204.
Janody, F., Lee, J., Jahren, N., Hazelett, D., Benlali, A., Miura, G.,
Draskovic, I. and Treisman, J. (2004). A mosaic genetic screen reveals
distinct roles for Trithorax and Polycomb group genes in Drosophila eye
development. Genetics 166, 187-200.
Kinzler, K. and Vogelstein, B. (1996). Lessons from hereditary colon cancer.
Cell 87, 159-170.
Lagna, G., Hata, A., Hemmati-Brivanlou, A. and Massagué, J. (1996).
Partnership between DPC4 and Smads in TGF-␤ signaling pathways. Nature
383, 832-836.
Lorson, C., Hahnen, E., Androphy, E. and Wirth, B. (1999). A single
nucleotide in the SMN gene regulates splicing and is responsible for spinal
muscular atrophy. Proc. Natl. Acad. Sci. USA 96, 6307-6311.
Marenda, D., Zraly, C. and Dingwall, A. (2004). The Drosophila Brahma
(SWI/SNF) chromatin remodeling complex exhibits cell-type specific
activation and repression functions. Dev. Biol. 267, 279-293.
Marquez, R., Singer, M., Takaesu, N., Waldrip, W., Kraytsberg, Y. and
Newfeld, S. (2001). Transgenic analysis of the Smad family of TGF-␤ signal
transducers in Drosophila suggests new roles and interactions between
family members. Genetics 157, 1639-1648.
Massagué, J., Blain, S. and Lo, R. (2000). TGF-␤ signaling in growth control,
cancer & heritable disorders. Cell 103, 295-309.
Newfeld, S., Chartoff, E., Graff, J., Melton, D. and Gelbart, W. (1996).
Mad encodes a conserved cytoplasmic protein required in DPP/TGF-␤
responsive cells. Development 122, 2099-2108.
Newfeld, S., Wisotzkey, R. and Kumar, S. (1999). Molecular evolution of a
developmental pathway: Phylogenetic analyses of TGF-␤ family ligands,
receptors and Smad signal transducers. Genetics 152, 783-795.
Nicholls, R. and Gelbart, W. (1998). Identification of chromosomal regions
involved in dpp function in Drosophila. Genetics 149, 203-215.
Nishita, M., Hashimoto, M., Ogata, S., Laurent, M., Ueno, N., Shibuya, H.
and Cho, K. (2000). Interaction between Wnt and TGF-␤ signaling
pathways during formation of Spemann’s organizer. Nature 403, 781-785.
Padgett, R., Wozney, J. and Gelbart, W. (1993). Human BMP sequences
confer normal dorsal-ventral patterning in Drosophila embryos. Proc. Natl.
Acad. Sci. USA 90, 2905-2909.
Peltomaki, P. (2001). Deficient DNA mismatch repair: a common etiologic
factor for colon cancer. Hum. Mol. Gen. 10, 735-740.
Pouliot, F. and LaBrie, C. (1999). Expression profile of agonistic Smads in
human breast cancer cells: absence of regulation by estrogens. Int. J. Cancer
81, 98-103.
Raftery, L., Twombly, V., Wharton, K. and Gelbart, W. (1995). Genetic
screens to identify elements of the dpp signaling pathway in Drosophila.
Genetics 139, 241-254.
Ray, R. and Wharton, K. (2001). Context-dependent relationship between
the BMPs gbb and dpp during development of the Drosophila wing disk.
Development 128, 3913-3925.
Riggins, G., Thiagalingam, S., Rozenblum, E., Weinstein, C., Kern, S.,
Hamilton, S., Willson, J., Markowitz, S., Kinzler, K. and Vogelstein, B.
(1996). Mad-related genes in the human. Nat. Genet. 13, 347-349.
Riggins, G., Kinzler, K., Vogelstein, B. and Thiagalingam, S. (1997).
Development
4894 Development 132 (21)
Frequency of Smad gene mutations in human cancers. Cancer Res. 57,
2578-2580.
Sampath, T., Rashka, K., Doctor, J., Tucker, R. and Hoffmann, F. (1993).
Drosophila TGF-␤ superfamily proteins induce endochondrial bone
formation in mammals. Proc. Natl. Acad. Sci. USA 90, 6004-6008.
Sansom, O., Reed, K., Hayes, A., Ireland, H., Brinkmann, H., Newton, I.,
Batlle, E., Simon-Assmann, P., Clevers, H., Nathke, I. et al. (2004). Loss
of APC in vivo immediately perturbs Wnt signaling, differentiation and
migration. Genes Dev. 18, 1385-1390.
Schutte, M., Hruban, R., Hedrick, L., Cho, K., Nadasdy, G., Weinstein,
C., Bova, G., Isaacs, W., Cairns, P., Nawroz, H. et al. (1996). DPC4 gene
in various tumor types. Cancer Res. 56, 2527-2530.
Sekelsky, J., Newfeld, S., Raftery, L., Chartoff, E. and Gelbart, W. (1995).
Genetic characterization and cloning of Mad, a gene required for dpp
function in Drosophila. Genetics 139, 1347-1358.
Shi, Y., Wang, Y., Jayaraman, L., Yang, H., Massagué, J. and Pavelitch,
N. (1998). Crystal structure of a Smad MH1 domain bound to DNA: insights
on DNA binding. Cell 94, 585-594.
Siegfried, E., Chou, T. and Perrimon, N. (1992). wg signaling acts through
zeste-white3, the Drosophila homolog of glycogen synthase kinase-3, to
regulate engrailed and establish cell fate. Cell 71, 1167-1179.
Skeath, J. and Carroll, S. (1991). Regulation of achaete-scute gene
expression and sensory organ pattern formation in the Drosophila wing.
Genes Dev. 5, 984-995.
Souza, R. (2001). A molecular rationale for the how, when and why of
colorectal cancer screening. Aliment. Pharmacol. Ther. 15, 451-462.
St Johnston, R., Hoffmann, F., Blackman, R., Segal, D., Grimaila, R.,
Padgett, R., Irick, H. and Gelbart, W. (1990). Molecular organization of
the dpp gene in Drosophila. Genes Dev. 4, 1114-1127.
Su, M., Wisotzkey, R. and Newfeld, S. (2001). A screen for modifiers of dpp
mutant phenotypes identifies lilliputian, the only member of the FragileX/Burkitt’s Lymphoma family of transcription factors in Drosophila.
Genetics 157, 717-725.
Thatcher, J., Haun, C. and Okkema, P. (1999). The DAF-3 Smad binds DNA
and represses gene expression in the Caenorhabditis elegans pharynx.
Development 126, 97-107.
Thiagalingam, S., Lengauer, D., Leach, F., Schutte, M., Hahn, S.,
Overhauser, J., Willson, J., Markowitz, S., Hamilton, S., Kern, S. et al.
(1996). Evaluation of candidate tumor suppressor genes on chromosome 18
in colorectal cancer. Nat. Genet. 13, 343-346.
Wisotzkey, R., Mehra, A., Sutherland, D., Dobens, L., Liu, X., Dohrmann,
C., Attisano, L. and Raftery, L. (1998). Medea is a Drosophila Smad4
homolog that is differentially required to potentiate Dpp responses.
Development 125, 1433-1445.
Wolff, T. (2000). Histological techniques for the Drosophila eye. Part I: Larva
and Pupa. In Drosophila Protocols (ed. W. Sullivan, M. Ashburner and R.
Hawley), pp. 201-227. New York: Cold Spring Harbor Laboratory Press.
Xu, J. and Attisano, L. (2000). Mutations in the tumor suppressors Smad2
and Smad4 inactivate TGF-␤ signaling by targeting Smads to the ubiquitinproteasome pathway. Proc. Natl. Acad. Sci. USA 97, 4820-4825.
Xu, X., Yin, Z., Hudson, J., Ferguson, E. and Frasch, M. (1998). Smad
proteins act in combination with synergistic and antagonistic regulators to
target Dpp responses to the Drosophila mesoderm. Genes Dev. 12, 23542370.
Yu, K., Sturtevant, M., Biehs, B., Francois, V., Padgett, R., Blackman, R.
and Bier, E. (1996). The Drosophila dpp and sog genes function
antagonistically during wing vein development. Development 122, 40334044.
Zhang, H., Chen, X., Wang, M., Wang, J., Qi, Q., Zhang, R., Xu, W., Fei,
Q., Wang, F., Cheng, Q., et al. (2004). Defective expression of TGF-␤
receptor type II is associated with CpG methylated promoter in primary nonsmall cell lung cancer. Clin. Cancer Res. 10, 2359-2367.
Research article