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First published online September 28, 2006
doi: 10.1242/10.1242/dev.02561
Department of Developmental and Cell Biology, and the Developmental Biology Center, University of California Irvine, Irvine, CA 92697, USA.
Authors for correspondence (e-mail:
karora{at}uci.edu;
rwarrior{at}uci.edu)
Accepted 3 August 2006
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SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Schnurri, Bone Morphogenetic Protein signaling, Bmp, Smad, Transcription, Transcriptional activation, Transcriptional repression, Evolutionary conservation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Massague et al.,
2005). Signaling is initiated by ligand binding to a complex of
type-I and type-II transmembrane serine/threonine kinase receptors, enabling
the type-II receptor to phosphorylate the type-I receptor. The type-I receptor
in turn phosphorylates a receptor-regulated member of the Smad (RSmad) family
of intracellular signal transducers. In vertebrates, Bmp signaling is mediated
by Smad1, Smad5 and Smad8, whereas in Drosophila the sole
Bmp-specific Smad is Mothers against dpp (Mad). The activated R-Smad
associates with the co-Smad, Smad4 [Medea (Med) in Drosophila],
forming a heteromeric complex that translocates into the nucleus. R-Smads and
co-Smads are structurally similar, possessing amino-terminal MH1 and
carboxyterminal MH2 domains separated by a flexible linker. A conserved
ß-hairpin motif in the MH1 domain mediates DNA binding, enabling the
R-Smad/Smad4 complex to direct transcriptional responses. Co-Smads recognize
the sequence GTCT (the `Smad-binding element' or SBE), whereas Smad1 and Mad
preferentially bind GCCGNC (Gao et al.,
2005; Johnson et al.,
1999; Kim et al.,
1997; Xu et al.,
1998). Smads bind to these low complexity sites with weak affinity
and it is thought that Smad-DNA interactions are enhanced by partnership with
other transcription factors that are crucial for promoter selectivity. In
support of this idea, reporter constructs containing multimers of SBEs or
Smad1 sites alone are incapable of replicating the expression patterns of
endogenous Bmp target genes in vivo, although they confer Bmp responsiveness
in cell culture (Shi and Massague,
2003; von Bubnoff et al.,
2005).
Drosophila Schnurri (Shn) is a large DNA-binding transcription
factor with multiple, widely separated zinc finger domains, and was one of the
first partners identified for Bmp-specific R-Smads
(Dai et al., 2000;
Udagawa et al., 2000). Shn
localizes to the nucleus and interacts with Mad and Med in response to Dpp
signaling. Genetic and phenotypical analysis has established that shn
is essential for Dpp signaling at many developmental stages and in diverse
tissues (Arora et al., 1995;
Grieder et al., 1995;
Staehling-Hampton et al.,
1995). At the molecular level, Shn uses two modes to regulate Dpp
target genes: transcriptional activation and repression. Shn can directly
activate a small subset of Dpp-responsive genes, but exerts most of its effect
through a relief-of-repression mechanism. Shn inhibits the expression of a
transcriptional repressor Brinker (Brk) that, in turn, negatively regulates
the expression of most Dpp target genes. Therefore, Dpp signaling leads to the
inhibition of brk expression and the derepression of batteries of
Dpp-responsive genes (Affolter et al.,
2001; Marty et al.,
2000; Torres-Vazquez et al.,
2001; Torres-Vazquez et al.,
2000). Shn represses brk via the formation of a
Shn/Mad/Med complex on defined elements in the brk promoter
(Muller et al., 2003;
Pyrowolakis et al., 2004).
Given the prominent role of Shn in Dpp signaling, an inevitable question is
whether the conservation encountered at other levels of the pathway extends to
the cis-regulatory elements and nuclear factors that mediate the
transcriptional response to Bmps. Furthermore, brk, a primary target
of Shn activity, is absent from genomes outside the arthropods, raising the
possibility that the involvement of Shn in Bmp signaling may be unique to
insects.
Unlike brk, Shn-related genes are found in other phyla. In C.
elegans the lone Shn homolog SMA-9 has been implicated in Bmp signaling,
although its mechanism of action is unknown
(Liang et al., 2003). Three
Shn homologs are found in vertebrates that also contain widely spaced, paired
zinc finger domains with high sequence similarity to the fly protein. Shn
proteins share additional features, including an unusually large size, overall
structural organization and the presence of acidic domains. Human members of
this family have been variously referred to as
Shn1/HIVEP1/MBP-1/PRDII-BF1/ZAS1, Shn2/HIVEP2/MBP-2/ZAS2 and
Shn3/HIVEP3/KRC/ZAS3 (Liang et al.,
2003; Wu, 2002).
No direct evidence ties vertebrate Shn1 or Shn3 to the Bmp pathway, but Shn2
has recently been shown to promote Bmp-responsive transcription of the
PPAR
gene in mouse embryonic fibroblasts. However, Shn2 acts through
sites that bear no resemblance to the sequences recognized by the
Drosophila Shn/Smad complex, and requires co-operativity with the
transcription factor C/EBP
(Jin et
al., 2006). Thus, whether the mechanism of action of Shn proteins
in Bmp signaling is phylogenetically conserved remains an unanswered
question.
Here, we demonstrate an unusual and unexpected conservation of the cis- and trans-regulatory elements involved in the response to Bmp signaling. We found that an element from the Xenopus Vent2 (Xvent2) promoter that confers sensitivity to Bmp signaling in vertebrate embryos also directs a Dpp-dependent response in Drosophila. However, although the element mediates transcriptional activation in Xenopus, it directs transcriptional repression in Drosophila. We demonstrate that the closest human Shn homolog, hShn1, can mediate signaling-dependent transcriptional activation through this element in vertebrate cells, implicating it in Bmp signal transduction. Remarkably hShn1 can also mediate signaling-dependent repression in Drosophila and rescues patterning defects in shn mutant embryos, whereas fly Shn can activate transcription in Xenopus assays. This switch in transcriptional output between repression and activation is not a function of differences in the cis-regulatory elements or an inherent property of the human and Drosophila proteins, indicating that Shn/Smad complexes are likely to recruit co-activators and co-repressors in a context-dependent manner.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Xvent2-BRE-lacZ contains five 23-bp Xvent2-BREs
between NotI and BamHI sites in Grh-lacZ. The 6.0
brk-lacZ transgene contains -8.3 to -2.7 kb of the brk
promoter driving lacZ (L.-C.Y., unpublished). All other stocks are
described in FlyBase. Embryos and imaginal discs were stained as described
(Torres-Vazquez et al.,
2000).
Rescue of shn mutants
Full-length Shn and hShn1 (Fan and
Maniatis, 1990) were subcloned into pUAST
(Brand and Perrimon, 1993).
shnP4738 is a null allele (Arora
et al., 1995). Progeny from UAS-Shn/UAS-hShn1,
shnP4738/CyO flies crossed to shnP4738/CyO; HS-Gal4
were aged to 8-10 hours and received two 1-hour heat shocks at 37°C,
separated by 25°C rest periods. brk expression was monitored
using the enhancer trap X-47
(Campbell and Tomlinson, 1999).
Whole-mount cuticles were prepared as described
(Arora et al., 1995).
Homozygous mutants were identified by the absence of wg-lacZ or
CyO, Kr>GFP.
Xenopus reporter gene analyses
pXvent2(-150)LUC and pXvent2-5xBRE(-150)LUC have
been described previously (von Bubnoff et
al., 2005). The oligonucleotide
5'-CGGCAGACAGGTTGGAGCCAGCTCGGCAGACAGGTTGGAGCCAGCT-3'
containing a BRE dimer (mutations underlined) was used to generate a PCR
product that was subcloned into pXvent2(-150)LUC to create
pXvent2-5xBRE(sub2)(-150)LUC.
pXvent2-6xBRE(del2)(-150)LUC was similarly created using
5'-CGGCAGAC_GGTGGAGCCAGCTCGGCAGAC_GGTGGAGCCAGCT-3 (underscores indicate
deleted bases). pXvent2-5xBRE(-150)GFP3,
pXvent2-5xBRE(sub2)(-150)GFP3 and
pXvent2-6xBRE(-150)(del2)GFP3 contain
BamHI-HindIII fragments from the respective reporter
plasmids in pCXGFP3 (a gift of Enrique Amaya, University of Manchester).
Full-length Shn and hShn1 were subcloned into pBSII-KS, containing the SV40
polyadenylation signal. Capped mRNAs were synthesized using the T3 mMessage
Machine Kit (Ambion), including 1 µl GTP/20 µl reaction, diluted into
single use `injection cocktails' containing reporter genes, stored at
-80°C and used within 24 hours.
mRNA concentrations/embryo were: luciferase reporters, 200 pg;
dominant-negative (Suzuki et al.,
1994) and constitutively active
(Candia et al., 1997) BmpR,
0.1-2.0 ng; and hShn1 and Shn, 2 ng. Wild-type and mutant BRE reporters were
microinjected into the animal poles of two- to four-cell stage embryos and
incubated until blastula stages 8-9. Mid-blastula stage animal caps were
dissected and homogenized for luciferase assays when sibling embryos reached
stages 10.5-11 (Watabe et al.,
1995). In situ hybridization and generation of transient
transgenic embryos using the sperm nuclear transfer technique have been
described previously (von Bubnoff et al.,
2005).
Biochemical assays
N-terminal FLAG-Mad and FLAG-Med, TkvA and ShnCT plasmids have been
described previously (Gao et al.,
2005). PCR fragments 1 (hShn11-599), 2
(hShn11-702), 3 (hShn1496-1121), 4
(hShn11002-1635), 5 (hShn11496-2213), 6
(hShn11756-2544) and 7 (hShn11756-2717) were subcloned
into the pAWM vector containing an Actin5C promoter and C-terminal 6xMyc
epitopes (a gift of T. D. Murphy, Carnegie Institution, Baltimore). Extracts
from Drosophila S2 cells transfected with individual hShn1 constructs
and TkvA/Mad/Med or TkvA/hSmad1/hSmad4 were incubated with anti-FLAG antisera.
Immunoprecipitates were run on 4-12% gradient SDS polyacrylamide gels before
western blotting and visualization of interacting protein with anti-Myc. Gel
shift assays were performed as described
(Gao et al., 2005;
Kirov et al., 1994;
Pyrowolakis et al., 2004).
Extracts from Drosophila S2 cells transfected with different plasmid
combinations were used to shift a Xvent2 BRE probe
(5'-CTAAGAGCTGGCTCCACCATGTCTGCCGTTAGTTGGCTCA-3').
For supershifts, 60 ng of anti-FLAG (M2, Sigma) or 100 ng of anti-MYC (9E10,
Santa Cruz) were added to the binding reactions. GST pull-downs were carried
out as described (Dai et al.,
2000). Human Smad1 and Smad4 linker+MH2 regions subcloned in
pGEX-4T-1 were expressed in E. coli BL21. PCR fragments of hShn1 were
subcloned into pCITE4A (Novagen) before in vitro translation using the TNT-T7
Coupled Transcription/Translation Kit (Promega).
Molecular phylogeny
Shn proteins used were: D. melanogaster DMU31368; X.
tropicalis Shn1 genome v4.1 gw1.33.114.1, Shn2 gw1.274.19.1, Shn3
ensemble.c_scaffold_ 478000040; Mouse Shn1 NM_007772, Shn2 NM_010437, Shn3
NM_010657; Human Shn1 NM_002114, Shn2 NM_006734, Shn3, NM_024503. CLUSTALW
analysis was performed using the San Diego Supercomputer Center's Biology
Workbench.
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RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Classical promoter analyses have identified a 53-bp Bmp responsive module
located at -191 relative to the initiator ATG
(Candia et al., 1997;
Hata et al., 2000;
Rastegar et al., 1999).
Phylogenetic footprinting and in vivo reporter gene analyses have further
refined the ciselement to a 23-bp BRE that is both necessary and sufficient
for Bmp-dependent activation in animal cap ectodermal explants and transgenic
embryos (von Bubnoff et al.,
2005). To test whether the 23-bp Xvent2-BRE can mediate
Bmp responsiveness in Drosophila, we generated transgenic lines
containing five tandem BREs upstream of lacZ in the 3xGrh
vector (henceforth referred to as Xvent2-BRE-lacZ;
Fig. 1A). As the Dpp pathway
utilizes both direct and indirect relief-of-repression mechanisms to activate
gene expression, we chose a single vector that would allow us to assay both
outputs. 3xGrh-lacZ contains binding sites for Grainyhead
(Grh), a transcriptional activator ubiquitously expressed in the ectoderm
after embryonic stage 11 (Bray and Kafatos,
1991; Furriols and Bray,
2001). Therefore, 3xGrh-lacZ is transcriptionally
silent until extended germ band stage, but then drives uniform lacZ
expression through later embryogenesis and in larval tissues
(Fig. 1B-E). Thus, enhancers
mediating activation can be assayed prior to stage 11, whereas elements
mediating repression can be detected at later stages.
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). Subsequently, dpp is involved in many other
aspects of embryonic and larval patterning
(Raftery and Sutherland,
1999). We analyzed Xvent2-BRE-lacZ transgenic
lines and found no reporter gene expression prior to stage 11, suggesting that
this element cannot direct Dpp-dependent activation in early embryos (data not
shown). However, from stage 11 onwards ß-galactosidase could be detected
in a distinct pattern in both embryos and imaginal discs
(Fig. 1F-I). Interestingly, the
reporter was downregulated where Dpp signaling is active
(Dorfman and Shilo, 2001;
Sutherland et al., 2003;
Tanimoto et al., 2000). In
stage 13 embryos (germband retraction), dpp is expressed in the
leading edge of the dorsal ectoderm and in the boundary between dorsal and
ventral ectoderm, both sites where reporter expression is absent (compare
Fig. 1F with 1N). Likewise,
lacZ is repressed in the medial cells of the wing imaginal disc, and
regions of eye-antennal and leg discs subject to Dpp signaling (compare
Fig. 1G-I with 1O-Q). At all
locations, the spatial and temporal pattern of reporter expression closely
replicates that of brk, a negatively regulated target of Dpp
signaling (Fig. 1J-M). These
remarkable parallels strongly suggest that the Xvent2-BRE, which
activates transcription in vertebrates, mediates transcriptional repression in
response to Bmp signaling in Drosophila.
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).
Repression elements in the brk promoter and several other direct Dpp
targets contain a GRCGNC(N5)GTCTG consensus, with binding sites for both Mad
and Med separated by a spacer of 5 bp (see
Fig. 2A). This precise spacing
is required for Shn to dock to Mad/Med complexes on the DNA and mediate
repression (Gao et al., 2005;
Pyrowolakis et al., 2004). A
comparison between the Xenopus BRE and the Drosophila
consensus sequence shows a near perfect match (see
Fig. 2A). Although this finding
suggested a simple explanation for the ability of the Xenopus BRE to
function in Drosophila, there is a single nucleotide difference
between the BRE and the Brk consensus (G
T at position 4) that has been
reported to prevent Mad/Med binding and formation of the repression complex in
flies (Pyrowolakis et al.,
2004). This change in a crucial residue raised the possibility
that the sequence similarity was purely coincidental and that the
BRE-dependent expression pattern arises through a mechanism that is
independent of Shn. To test whether BRE function requires Shn, we examined
Xvent2-BRE-lacZ expression in shn-
embryos. Xvent2-BRE-lacZ is derepressed throughout the
ectoderm from stage 11 onwards, indicating that Shn is crucial for restricted
BRE expression (data not shown). Next, we tested whether the Xenopus
BRE can nucleate assembly of a Shn/Mad/Med complex required for repression.
Drosophila S2 cells were transfected with constitutively activated
Dpp type-I receptor Thickveins (TkvA), epitope-tagged Mad, Med, and a
C-terminal fragment of Shn (ShnCT) that has been shown to participate in
complex formation (Pyrowolakis et al.,
2004). Radiolabelled BRE oligonucleotides were incubated with cell
extracts and subjected to gel electrophoresis. We found that Mad and Med
decreased the mobility of the BRE fragment
(Fig. 1R, lane 2), and addition
of Shn further decreased its migration
(Fig. 1R, lane 4). Furthermore,
both Mad/Med and the Shn/Mad/Med complexes could be supershifted by antisera
against FLAG and Myc epitopes on the Smads and Shn, respectively (compare lane
2 with 3, and 4 with 5), confirming the presence of these proteins in the
shifted bands. The ability of the Xvent2-BRE to assemble a
Shn/Mad/Med complex despite the difference in sequence between the
Drosophila and Xenopus elements may be due to compensatory
effects from adjacent or flanking residues. This biochemical analysis
indicates that the Xvent2-BRE can mediate Bmp signaling-dependent
transcriptional repression through a mechanism similar to that used by the
brk element.
The Xenopus and Drosophila cis-elements share architecture critical for Bmp responsiveness
The similarities between the Xenopus BRE and the Drosophila
brk element, both in sequence and in the ability to assemble a
signaling-dependent repression complex, raise an intriguing question. Do these
parallels reflect an underlying conservation of the Bmp-responsive
transcriptional regulators? To address this issue, we first tested whether
mutations that abolish repression by the brk element in
Drosophila correspondingly alter the ability of Xvent2-BRE
to respond to Bmp signaling in Xenopus. Substitutions of individual
residues in the Mad and Med sites of the brk element interfere with
complex formation and affect repression in vivo
(Pyrowolakis et al., 2004). In
previous studies, we and others have shown that Smad4 binds the BRE and that
mutations that compromise the stimulation of Xvent2 reporters map to
regions corresponding to the Smad sites
(Hata et al., 2000;
Henningfeld et al., 2000;
Karaulanov et al., 2004;
von Bubnoff et al., 2005). A
distinguishing feature of the Drosophila element is the requirement
for an invariant spacing between the Mad and Med sites that is crucial for the
recruitment of Shn to the Smad-DNA complex
(Pyrowolakis et al., 2004). If
a similar complex binds the Xenopus BRE, the spacer sequences could
serve as docking sites for a Smad co-factor, such as a vertebrate Shn (see
Fig. 1R). We therefore
generated BRE-luciferase reporters that alter either the length or the
sequence of the Xvent2 spacer and tested their ability to respond to
Bmp signaling in Xenopus animal cap assays
(Fig. 2B, see Materials and
methods). The wild-type Xvent2-BRE drives high-level luciferase
expression in response to endogenous Bmps that is further increased upon
co-injection of a constitutively active Bmp receptor (CABR), demonstrating
that the reporter responds to changes in activity of the Bmp pathway
(Fig. 2C). Substitution of 2 bp
within the spacer without altering its length (Xvent2-sub2), does not
affect Bmp responsiveness (Fig.
2A,C). By contrast, shortening the spacer by deleting 2 bp
(Xvent2-del2) rendered the reporter insensitive to stimulation
(Fig. 2A,C). These results
demonstrate that the Xvent2-BRE responds to Bmp stimulation in a
manner identical to that observed for the brk element in
Drosophila.
To examine whether the response to endogenous Bmp signaling places similar
constraints on BRE architecture, we assessed the activity of these constructs
in transgenic Xenopus (Kroll and
Amaya, 1996; von Bubnoff et
al., 2005). Wild-type and mutant BRE multimers were cloned
upstream of the Xvent2 minimal promoter driving GFP, and reporter
gene expression was examined by in situ hybridization. The wild-type BRE
drives expression at several sites where Bmp4 is transcribed, including the
eye, otic vesicle, ventral branchial arches, and the proctodeal region
(Fig. 2C)
(Karaulanov et al., 2004;
von Bubnoff et al., 2005). The
Xvent2-sub2 mutant drives a similar expression pattern. By contrast,
transgenic embryos carrying the Xvent2-del2 mutant show random
expression patterns, presumably as a result of integration site-dependent
position effects. These data reinforce the idea that in vivo (as in animal cap
assays) maintenance of an invariant spacer length is crucial for the BRE to
respond positively to Bmp signaling in Xenopus. Thus, BRE function
has the same architectural requirements in Xenopus and
Drosophila, and this constraint on the organization of the element
suggests that the recruitment of a Smad-interacting protein analogous to Shn
could be essential for BRE activity in vertebrates.
Shn proteins stimulate BRE activity in vertebrate cells
Shn proteins have high homology in the paired C2H2 zinc finger domains but
show limited sequence identity outside of these regions
(Fig. 3A) (reviewed by
Wu, 2002). Generation of a
molecular phylogeny of fly, worm, frog, mouse and human proteins indicates
that Drosophila Shn is most closely related to vertebrate Shn1
(Fig. 3B). Therefore, to
investigate whether Shn proteins play mechanistically similar roles in the Bmp
pathway, we examined whether human Shn1 (hShn1) could stimulate the
BRE-mediated response to Bmp signaling in animal cap assays
(Fig. 3C). We found that
expression of hShn1 alone resulted in an approximately 4-fold stimulation of
the reporter that was further enhanced to 16-fold in the presence of an
activated Bmp receptor. This result suggests that vertebrate Shn-related
proteins could play an important role in mediating Bmp signaling.
Because the Xenopus BRE was Dpp responsive in Drosophila embryos, we were interested in determining whether Drosophila Shn can also elicit a response in Xenopus animal caps. We found that Shn alone did not significantly stimulate the reporter (Fig. 3D). Remarkably, co-injection of Shn and activated Bmp receptor significantly elevated reporter activity (Fig. 3D). Because fly Shn is capable of participating in a Smad complex on the Xvent2-BRE in vitro (see Fig. 1R), these data suggest that Shn can function as a transcriptional activator in Xenopus embryos. This result is striking as, in Drosophila, Shn mediates repression via the same cis-element. This difference in modality suggests that whether Shn drives transcriptional activation or repression is context-dependent rather than an inherent property of the protein.
Vertebrate Shn interacts directly with Smad proteins and assembles a complex on the BRE
The functional similarities between hShn1 and Shn suggest that vertebrate
Shn proteins act as Smad co-factors on the Vent2 BRE. However docking
of Shn to the Mad/Med complex requires zinc fingers 6 and 8
(Pyrowolakis et al., 2004),
which are not represented in vertebrate Shn proteins (see
Fig. 3A). However, we, and
others, have shown that Drosophila Shn contains at least two
additional Smad-interacting domains (Dai et
al., 2000; Udagawa et al.,
2000), raising the possibility that interactions through other
regions contribute to hShn1-Smad complex formation on the Xvent2-BRE.
We therefore sought to identify and localize potential Smad-interacting
regions within hShn1 by co-expressing hShn1 fragments with hSmad1 and an
activated Bmp receptor. Co-immunoprecipitation assays identified two separate
domains (hShn11-599 and hShn11756-2544) that mediate
Smad interactions (Fig. 4A).
The ability of hShn1 to bind hSmad1 was independently confirmed in GST
pull-down assays. As demonstrated in Fig.
4B, fragments hShn11-702 and hShn11756-2544
bind GST-Smad1 strongly and GST-Smad4 with lower affinity, suggesting that
Shn/Smad interactions are conserved in vertebrate proteins. To determine
whether hShn1 binding to Smads results in the formation of a DNA-protein
complex on the Xvent2-BRE, different hShn1 polypeptides were
subjected to gel shift analysis using Drosophila Smads. We found that
hShn11-599 and hShn11496-2213 could bind and supershift
a BRE oligonucleotide (Fig.
4A,C, lanes 4 and 6). These polypeptides correlate well with
fragments that tested positive in the co-immunoprecipitation and GST pull-down
experiments, and suggest that the Smad1-interaction domains are located in the
region of overlap. Collectively, these data strongly suggest that, despite the
difference in modality, vertebrate Shn proteins use a conserved molecular
mechanism to mediate the transcriptional response to Bmp signaling.
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DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Padgett et al., 1993;
Sampath et al., 1993). In this
study, we show that both Drosophila Shn and its vertebrate homolog
Shn1 can use a common mechanism involving interaction with Smads and similar
cis-regulatory elements to transduce Bmp signals. Another important finding is
that Shn and hShn1 can mediate either transcriptional activation or repression
through identical cis-elements, depending on the cellular context. These
results demonstrate that Shn proteins are not obligate transcriptional
repressors but rather that they recruit co-activators or co-repressors in a
cell-type specific fashion.
The brk element is a phylogenetically conserved cis-regulatory motif
Our data indicate that a common set of simple parameters - the presence of
Smad1 and Smad4 binding sites with a 5-bp separation - govern the ability of
the Xvent2-BRE to respond to Bmp signaling in both vertebrate cells
and in Drosophila. We establish that the BRE can assemble a Smad/Shn
complex despite a GT substitution at position 4 (see
Fig. 2A). This was unexpected,
as the change affects one of the two GNCN sites in the brk consensus
site that are thought to bind Mad (Gao and
Laughon, 2006; Gao et al.,
2005). One possibility is that protein-protein interactions render
the presence of both GNCN sites unnecessary. Additionally, it has recently
been shown that an AC substitution at position 5 in the brk
element enhances Mad binding (Gao and
Laughon, 2006). Interestingly the Xvent2-BRE also
contains a C at this position that could play a compensatory role and aid
complex formation. Thus, our study suggests a modified consensus recognition
site GRCKNC(N5)GTCTG for the Shn/Smad complex. The conservation of both
sequence and function in the BRE is unexpected. For example, even
skipped (eve) stripe 2 enhancers from the related species D.
yakuba and D. melanogaster, which drive expression in similar
patterns and are functionally equivalent, show considerable sequence
divergence, suggesting that `turnover' in transcription factor binding sites
and their relative spacing is likely to be common, with co-evolutionary
changes being necessary to retain essential functions
(Ludwig, 2002;
Tautz, 2000). The stability of
the Shn/Smad site differs from the eve element and is reminiscent of
the cross-phyla conservation of enhancers in the Dorsal/NF
B and wound
healing pathways in Drosophila and vertebrates
(Gonzalez-Crespo and Levine,
1994; Mace et al.,
2005).
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).
The extent to which the BRE confers Bmp responsiveness in vertebrates remains
to be established. In addition to Xvent2, this element is precisely
retained in the promoters of the related Xvent1B and Xvent2B
genes, as well as in the human and zebrafish homologs VENTX2, Vox and
Vent. The BRE consensus is also present in the Bmp-responsive
enhancers of the id1, id2, id3 and id4 genes
(Karaulanov et al., 2004;
von Bubnoff et al., 2005).
These elements in the id genes are also conserved between humans,
rats, mice, fish and frogs, which strongly suggests that the BRE represents an
ancient and functionally important motif. Limited searches have identified
BRE-like sequences in several other Bmp-responsive genes (Q. Zeng and
K.W.Y.C., unpublished), although more extensive bioinformatics combined with
in vivo verification will be required to gauge the full spectrum of genes
regulated by Shn proteins. It is likely that Shn proteins regulate only a
subset of Bmp targets through BRE-like motifs, as close matches to the
consensus are absent from several characterized Bmp-responsive promoters. This
is not unexpected, as an important function of Smad-interacting cofactors
would be the provision of cell, tissue and promoter specificity.
A previous study has implicated another Smad-interacting factor, OAZ, in
activation of Xvent2 transcription
(Hata et al., 2000). Although
it remains possible that OAZ may contribute to Xvent2 expression in
some tissues, the OAZ expression pattern is insufficient to explain all
aspects of Xvent2 synexpression with Bmps. In addition, the OAZ site
GCTCCA (that overlaps with the GRCKNC Smad1 motif) was retained in the
Xvent2-del2 mutation that abolished Bmp responsiveness in animal caps
and embryos (see Fig. 2), which
suggests that OAZ may not have a crucial role in Xvent2
regulation.
Shn proteins display context-dependent modalities
A striking feature of the Shn complex assembled at the BRE is that it leads
to opposite outputs in Drosophila and Xenopus. In
Drosophila, the brk element appears to exclusively mediate
repression both in cell culture and in vivo
(Gao et al., 2005;
Muller et al., 2003).
Repression is dependent upon recruitment of Shn to a Mad/Med complex, and this
suggested that Shn might function as an obligate repressor
(Pyrowolakis et al., 2004).
Consistent with this view, a 1-bp increase or decrease in the brk
spacer prevents Shn recruitment and changes the response from repression to
activation in cell culture assays (Gao et
al., 2005). The results presented in this work do not fit such a
model. Although Shn/Mad/Med bound to the Xenopus BRE mediates
repression in Drosophila (see Fig.
1), in Xenopus Shn elicits activation through the same
response element (see Fig. 3D).
Thus the crucial determinant of whether the complex mediates activation or
repression appears to be the cellular milieu rather than the presence of Shn.
Human Shn1 similarly behaves as a transcriptional repressor in flies and as an
activator in Xenopus (see Fig.
3B, Fig. 4) further
emphasizing the fact that the transcriptional polarity of Shn proteins may
depend on differential interaction with co-repressors and co-activators
(Fig. 6). Furthermore, there is
evidence that Drosophila Shn need not be an obligate repressor in
flies. We have previously shown that Shn can directly stimulate the expression
of Ubx, a Dpp target gene, both in vivo and in cell culture
(Dai et al., 2000) (see
Fig. 6). Likewise, Shn proteins
may not be obligate activators in vertebrate cells. Mouse Shn1 represses
expression of collagen21 through the recruitment of SPEN, a protein
that interacts with co-repressors SMRT/NCoR and HDAC
(Yang et al., 2005). Thus, the
outcome of Shn interactions with the BRE may not be a species-specific
phenomenon. Instead, our observations suggest that the modality of Shn is
regulated in a celltype specific manner. This has several ramifications.
First, it identifies Shn co-activators and co-repressors as additional, and as
yet uncharacterized, players in the Bmp pathway. Second, the ability of human
Shn proteins to repress genes in Drosophila leads us to speculate
that at least some of the co-repressors are evolutionarily conserved, and that
vertebrate Shn proteins may also be involved in the downregulation of Bmp
targets.
In Shn, zinc fingers 6 and 8 that mediate complex formation with Mad/Med
and an adjacent 113 amino acid domain are essential for repression
(Pyrowolakis et al., 2004).
Surprisingly, hShn1 that does not contain either the Smad interaction domain
or the `repression domain' can nevertheless effectively repress
brk-lacZ expression in flies (see
Fig. 5). As we have
demonstrated, hShn1 can assemble a complex on the BRE through two independent
Smad-interacting regions (see Fig.
4C), thus it appears that co-repressor recruitment may depend on
structural motifs/charge interactions that are not apparent from the primary
sequence.
Shn function in vertebrate Bmp signaling
Our results show that Shn1 can transduce a Bmp signal in Xenopus
and Drosophila embryos, as well as in cell culture (see Figs
3,
5; S.P. and R.W., unpublished).
The transcript distribution of Shn1 in Xenopus has
significant overlap with, and could explain, the Bmp-dependent synexpression
pattern of the Xvent2 gene from which the BRE was derived
(Durr et al., 2004;
Niehrs and Pollet, 1999;
Onichtchouk et al., 1996).
However, a complete understanding of the role of Shn proteins in vertebrates
will require loss-of-function analyses. Recent studies of knockouts for the
closely related Shn2 gene has shown that it contributes to
Bmp-responsive transcriptional activation of PPAR2, a key regulator of
adipocyte differentiation. Consistent with this, although Shn2 null
mice are viable and fertile, they have reduced adipose tissue. Interestingly,
although Shn2 also forms a complex with Smad1/Smad4 in response to Bmp
signaling, it mediates transcriptional activation via a distinct mechanism
(Jin et al., 2006). The
sequence recognized by the complex does not resemble the BRE and in fact lacks
a Smad1 site. Instead, activation of PPAR2 expression by Shn2 requires
sites for Smad4 and the transcription factor C/EBP- (see
Fig. 6). Mice lacking
Shn3 are also viable and fertile, but they show defects in immune
function. Shn3 has been shown to act downstream of the T cell and Tnf
receptors (Oukka et al., 2002;
Oukka et al., 2004), but
whether these events are linked to Bmp signaling remains to be explored.
All three vertebrate Shn proteins contain the zinc finger domains
implicated in Smad interaction
(Pyrowolakis et al., 2004;
Jin et al., 2006) (this work),
raising questions about how related Shn proteins contribute to Bmp signaling.
One possibility is that all Shn proteins can interact with Smads but that each
paralog recruits different sets of cofactors. Thus, each Shn protein would
regulate a distinct subset of targets, allowing Bmp signaling to control a
wide range of target genes in a selective manner. Alternatively, vertebrate
Shn proteins may share the ability to interact with common co-factors and have
redundant or partially overlapping functions. In light of this, it is
interesting that Shn2 and Shn3 mutant mice are viable, in
contrast to the embryonic lethality of mutations in Bmp ligands, receptors and
Smads (reviewed by Zhao,
2003). Mouse Shn1 and Shn2 are widely expressed
in overlapping tissues during embryogenesis and in adults, whereas
Shn3 shows restricted expression, suggesting that it may have a more
limited role (reviewed by Wu,
2002). A more complete understanding of how Shn proteins
contribute to Bmp signaling in mice will require the analysis of mouse
Shn1 knockouts, as well as the generation of double and triple null
animals. Our attempts to knockdown Shn1 function in X. tropicalis
using splice-site directed morpholino oligonucleotides have been unsuccessful
(I.L.B. and K.W.Y.C., unpublished). Given that all three genes are
ubiquitously distributed in gastrula stage embryos and continue to be
expressed throughout early development
(Durr et al., 2004), one
possibility is that vertebrate Shn proteins are functionally redundant.
Alternatively, because the morpholinos do not target mature mRNA, the absence
of a phenotype may be due to perdurance of the maternal message. The
conservation of the size and organization of Shn-like proteins suggests that
their structural properties may contribute to their function. One possibility
is that they act as molecular scaffolds for the assembly of the
transcriptional machinery that mediates Bmp responsiveness (see
Fig. 6). Shn proteins could
also serve an architectural function by promoting bending or looping of the
DNA, by binding at more than one location.
|
ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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10- to 11-fold in response to CABR, compared with reporter
alone. By contrast, Xvent2-del2 bearing a two-nucleotide spacer
deletion (see A) fails to respond. In the experiment shown, 100 pg of CABR
mRNA/embryo was used to stimulate expression; however, the
Xvent2-del2 reporter fails to respond even at 2 ng. All results are
presented as fold activation relative to wild type in the absence of CABR. We
consistently observed higher luciferase counts for Xvent2-sub2
relative to wild type. (C, right) Wild type and Xvent2-sub2 respond
to endogenous Bmp signaling, whereas the Xvent2-del2 mutant does not.
Transgenic embryos containing BRE multimers driving GFP are at tailbud stage
31/32. In situ hybridization shows that wild type and Xvent2-sub2
mutants direct expression in a pattern similar to the endogenous
Xvent2 mRNA and Xvent2 transgenes described previously.
Transgenic Xvent2-del2 embryos had variable expression patterns that
are likely to reflect position effects due to random integration sites.
