![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 12, No. 24, pp. 3900-3909, December 15, 1998
1 Howard Hughes Medical Institute and Laboratory of Molecular Biology, and 2 Laboratory of Genetics, University of Wisconsin, Madison, Wisconsin 53706 USA; 3 Institute for Molecular Biology and Genetics, Seoul National University, Kwanak-Ku, Seoul, 151-742, Korea
 |
Abstract |
|---|
 Top
 Abstract
 Introduction
Results
Discussion
Materials and methods
References
|
|---|
A small number of major regulatory (selector) genes have been identified in animals that control the development of particular organs or complex structures. In Drosophila, the vestigial gene is required for wing formation and is able to induce wing-like outgrowths on other structures. However, the molecular function of the nuclear Vestigial protein, which bears no informative similarities to other proteins, was unknown. Here, we show that Vestigial requires the function of the Scalloped protein, a member of the TEA family of transcriptional regulators, to directly activate the expression of genes involved in wing morphogenesis. Genetic and molecular analyses reveal that Vestigial regulates wing identity by forming a complex with the Scalloped protein that binds sequence specifically to essential sites in wing-specific enhancers. These enhancers also require the direct inputs of signaling pathways, and the response of an enhancer can be switched to another pathway through changes in signal-transducer binding sites. Combinatorial regulation by selector proteins and signal transducers is likely to be a general feature of the tissue-specific control of gene expression during organogenesis.
[Key Words: Organogenesis; signal transduction; selector gene; cis-regulatory elements; wing development]
| |
Introduction |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
Induction of gene expression by signaling molecules is a central mechanism controlling the patterning of developmental fields. Several short- and long-range signaling proteins have been identified and it has become increasingly clear that their signaling pathways are required for the development of most body structures. However, we know little about the molecular mechanisms that govern the competence of a target tissue to respond to a signal in a particular way. This raises the question of the identity and function of regulatory molecules that may work in conjunction with common signaling pathways to activate tissue-specific patterns of gene expression.
The development of the Drosophila wing is organized by two
orthogonal systems of short- and long-range signaling proteins that
regulate growth and gene expression (Brook et al. 1996
; Neumann and
Cohen 1997; Serrano and O'Farrell 1997). The Decapentaplegic (Dpp) and
Wingless (Wg) long-range signaling proteins are produced by cells along
the anteroposterior and dorsoventral compartment boundaries, and induce
the expression of several target genes in the developing wing disc. Dpp
induces the spalt (sal), spalt related
(salr), and optomotor blind (omb) genes in a
nested pattern centered on the Dpp stripe (de Celis et al. 1996a; Grimm
and Pflugfelder 1996; Lecuit et al. 1996; Nellen et al. 1996;
Sturtevant et al. 1997). The vestigial (vg) gene is
even more broadly induced by Dpp and its expression pattern fills the
entire wing field (Williams et al. 1991, 1993; Kim et al. 1996).
Activation of vg also requires input from the dorsoventral
organizing signals Wg and Serrate (Ser). Similarly, the Serum
Response Factor (SRF or blistered) gene is
regulated by signals from both axes and is expressed in the subregions
of the developing wing that become the intervein tissue (Montagne et
al. 1996). Other genes have expression patterns that are determined by
the signals that organize the dorsoventral axis, for example
cut is expressed in a stripe along the dorsoventral compartment boundary and regulated by Notch signaling (de Celis et al.
1996b; Neumann and Cohen 1996; Micchelli et al. 1997).
Recent studies have shown that the expression patterns of these target
genes in the wing field are controlled by discrete cis-regulatory elements that respond to particular signaling
proteins. Activation of vg is regulated by two separate
enhancers that are induced by different signaling pathways. The
boundary enhancer is activated along the dorsoventral boundary through
direct input of the Notch (N) pathway (Williams et al. 1994; Kim et al.
1996), whereas the quadrant enhancer is activated in the remainder of the growing wing pouch by Dpp and Wg signaling (Kim et al. 1996, 1997;
Zecca et al. 1996; Neumann and Cohen 1997). Similarly, the SRF
gene has two enhancers, one expressed in the intervein C region along
the anteroposterior compartment boundary that is regulated by Hh, and
one expressed in adjacent intervein regions (U. Nussbaumer, J. Montagne, J. Groppe, and M. Affolter, in prep.). Finally, enhancers were identified that drive sal and cut expression in
the wing pouch (Jack et al. 1991; Kuhnlein et al. 1997). Importantly,
all six enhancers are expressed exclusively in the wing (and haltere) pouch, and are not active in leg or eye-antennal discs, even though the
inducing signals are active in other tissues.
How are the cut, sal, SRF, and vg
enhancers induced specifically in the developing wing pouch but not in
other tissues in which these signaling pathways are also active?
Apparently, other factors must exist that promote or restrict the
responses of cells to signaling inputs. One candidate for such a role
is the Vg protein because it is able to reprogram some cells in leg and
other imaginal discs to adopt a wing fate (Kim et al. 1996). Vg is
required in all wing cells and loss of Vg function eliminates wing
formation (Williams et al. 1991, 1993). Vg is expressed in the
developing wing discs, but not in leg or eye antennal imaginal discs.
Thus, Vg is a selector protein for wing identity and development.
However, the molecular function of the Vg protein is not known,
although its nuclear localization (Williams et al. 1991) suggests that it could be involved in regulating gene expression.
Here we show that Vg induces wing development and wing-specific gene
expression by cooperating with the Scalloped (Sd) protein, a member of
the TEA family of DNA-binding proteins (for review, see Jacquemin and
Davidson 1997) that is also expressed in the developing wing pouch
(Williams et al. 1991, 1993; Campbell et al. 1992). We found that Sd
function is required in parallel to Vg to induce wing development.
Moreover, Sd and Vg act synergistically to induce several wing-specific
enhancers in tissue culture cells. A functional analysis of the
SRF intervein C enhancer revealed that this element contains
several binding sites for Sd that are essential for enhancer activity
in tissue culture and in vivo. Our results, together with the finding
that Vg and Sd interact physically (Simmonds et al. 1998), support a
model in which Vg and Sd form a complex that directly regulates gene
expression and limits the induction of their target enhancers to the
developing wing. Thus, it appears that the combinatorial control of
cis-regulatory elements by signal transducers and selector
proteins is an important mechanism to evoke tissue-specific responses
to common signals.
| |
Results |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
Vg cell autonomously induces wing-specific gene expression
Because ectopic expression of Vg in many imaginal discs induces the
outgrowth of wing tissue (Kim et al. 1996), we examined whether the
expression of various wing patterning genes was induced in these
ectopic growths. Vg is expressed in the entire developing wing pouch
(Fig. 1A) whereas Sal and SRF have specific
expression patterns within this domain (Fig. 1B,C) but are not
expressed in wild-type leg discs (Fig. 1D). Targeted expression of Vg
with the Gal4-UAS system induced ectopic expression of Sal and SRF in
developing leg imaginal discs (Fig. 1E,F) (Weatherbee et al. 1998).
Similarly, the nubbin (nub) gene, which is also
expressed and required during wing development (Ng et al. 1995), was
ectopically induced in leg discs by Vg expression (Klein and Martinez
Arias 1998; data not shown). In each case, only a subset of the cells expressing Vg activated the target gene, which suggests that additional factors control the expression pattern of each gene.
|
In a first step toward elucidating the molecular mechanism by which Vg regulates gene expression, we studied the response of wing-specific enhancers to ectopic Vg expression. We focused our attention on the boundary and quadrant enhancers of the vg gene (Fig. 2A,D) and the enhancer from the SRF gene that drives expression specifically in the intervein region between veins three and four (intervein C, Fig. 2G) (U. Nussbaumer, J. Montagne, J. Groppe, and M. Affolter, in prep.). All three enhancers were induced by ectopic Vg expression in leg and other imaginal discs. Importantly, ectopic expression of Vg in clones of cells induced the enhancers only within the clones (Fig. 2B,C,E,F,H,I). However, gene expression was not induced in all cells within clones nor in all clones. In addition, each individual enhancer was expressed in different regions of these discs that appear to correlate with the spatial distribution of the different signaling inputs known to be required for activation of these enhancers.
|
Sd is required in parallel for Vg function
In the notum primordia of the wing disc, the vg enhancers,
as well as the sal, SRF, and nub genes were
not induced by ectopic Vg even though the known required extracellular
signals were present (Fig. 3A-C; data not shown).
Target gene activation could depend then on the function of another
gene(s). One candidate for such a factor is the product of the
sd gene, which is expressed in a pattern similar to Vg in the
wing disc (Fig. 3D) (Campbell et al. 1992) and is required for wing
formation (Campbell et al. 1991; Williams et al. 1993) and the proper
expression of Vg (Williams et al. 1993) and other genes (Morcillo et
al. 1996). In other discs, such as the leg and eye discs, sd
is endogenously expressed and is upregulated wherever ectopic Vg was
able to induce wing-specific gene expression and trigger wing
development (data not shown). We note, however, that a sd
enhancer trap line and the SRF-intervein C enhancer transgene were also
ectopically induced by Vg in the presumptive notum, although at levels
lower than those observed in the wing pouch (Fig. 3E,F). This is
consistent with the inability of Vg to trigger wing development and
induce other wing patterning genes in the developing notum. On the
basis of the correlation between sd expression and activation
of Vg target genes, we investigated whether Sd function was required in
parallel to Vg to exert its wing inducing activity.
|
To determine if Sd is required in vivo for Vg to induce the expression of wing patterning genes, we first examined expression of target genes in clones that were homozygous for a strong mutation in the sd gene. Expression of the SRF and vg genes was lost in these clones (Fig. 3G,H). The loss of SRF gene expression could have been due directly to the loss of Sd or indirectly to Sd effects on vg expression. To distinguish between these possibilities, we generated sd mutant clones in imaginal discs in which Vg was ectopically expressed via the Gal4-UAS system, thus bypassing the requirement of Sd for vg expression. In these sd mutant clones, SRF (Fig. 3J-L) and vg quadrant enhancer expression (Fig. 3I) was not induced, revealing that Sd function is required for Vg to regulate gene expression.
To distinguish whether Sd was required downstream of or in parallel to
Vg, we provided Sd activity in conjunction with Vg in ectopic
expression experiments. High levels of ectopic Sd expression produced
by the Gal4-UAS system with a dpp-GAL4 driver had a
dominant-negative effect, suppressing the ectopic wing phenotypes
caused by Vg expression, and repressing endogenous Sal expression in
the wing disc (not shown). It appears then that the concentration of Sd
is critical for gene expression, a parameter that is also critical for
target gene activation in tissue culture by Sd (see below) and the
human Sd homolog transcriptional enhancer factor-1 (TEF-1) (Xiao et al.
1991; Hwang et al. 1993). To better control the level of Sd expression,
we used a heat-inducible transgene. Coexpression of Sd (by heat shock)
and Vg (with a dpp-Gal4 driver) resulted in ectopic
outgrowths in the notum and induction of Sal expression along the
entire A/P boundary and SRF expression within the ectopic outgrowth (Fig. 3M-O). These phenotypes were never observed by expressing Vg or Sd alone. We conclude that Sd and Vg are required in
parallel to induce wing development.
Vg and Sd act synergistically to activate target genes
These genetic experiments, and the discovery that the Vg and Sd
proteins interact physically (Simmonds et al. 1998), led us to
investigate whether the activation of target genes might occur through
a direct interaction between Vg and Sd and cis-regulatory sequences of target genes. We found that the three wing-specific enhancers from the SRF and vg genes were activated
synergistically when Sd and Vg were coexpressed in Drosophila
S2 cells (Fig. 4). Although each individual protein
had some effect on reporter gene expression, this was always
significantly less than that observed in the presence of both Vg and Sd
(Fig. 4). Titration of the amounts of transfected Vg and Sd plasmids
with all enhancers showed that the relative concentration of the two
factors was critical and, at any given Vg concentration, high levels of
Sd reduced activation (Fig. 4B).
|
To define the sequences of the enhancers that respond to Vg/Sd, we analyzed the activation of smaller fragments from the 704-bp SRF intervein C enhancer, the 806-bp vg quadrant enhancer, and the 754-bp vg boundary enhancer in tissue culture (Fig. 4A). We found that a 125-bp fragment (SRF-A) derived from the 5' end of the SRF enhancer was activated, whereas an adjacent 131-bp fragment (SRF-B) was not activated (Fig. 4C). A 65-bp fragment from the vg quadrant enhancer (MD2) has been identified that, when multimerized, produces an expression pattern very similar to the full-length enhancer in wing discs (A. Hudson and S. Carroll, unpubl.). When assayed in tissue culture, MD2 was activated by Vg and Sd (Fig. 4D). Within the vg boundary enhancer, a 120-bp fragment sufficient to drive reporter gene expression in the wing pouch (vg-A) as well as a nonoverlapping 90-bp fragment (vg-B) were also activated synergistically by cotransfection of Vg and Sd (Fig. 4E).
Sd affects Vg localization to nuclei
One possible reason for the importance of the concentration of Sd on
Vg function concerns the localization of the Vg protein. We observed
that in S2 cells transfected with the vg expression plasmid
alone, the Vg protein appears to be localized to both the cytoplasm and
the nucleus (Fig. 5A-C). In contrast, in cells cotransfected with the Vg and Sd expression plasmids, Vg is clearly localized to nuclei (Fig. 5D-F). Furthermore, we note that Vg localization is more diffuse in sd mutant clones than in
sd+ cells; this is also true of ectopic Vg localization in
regions of imaginal discs that lack endogenous Sd expression (not
shown). Furthermore, deletion of the Sd interaction domain of Vg
results in cytoplasmic accumulation of Vg in vivo (Simmonds et al.
1998). Thus, Sd may facilitate the transport or retention of Vg protein in the nucleus and, coupled with the concentration-dependent, synergistic effects of Vg and Sd on target gene expression, these results suggest that the proteins form a complex in vivo.
|
Sd binds specifically to essential sites for target gene activation
Because Sd is a member of the TEA family of transcriptional
regulators that contain the highly conserved TEA DNA-binding domain (Jacquemin and Davidson 1997; Davidson et al. 1988), we investigated whether Sd could bind to the regulatory elements of the SRF
and vg genes in vitro. We used purified TEA domains in gel
mobility shift experiments, as the TEA domain alone is able to bind to DNA cooperatively and with high affinity (Hwang et al. 1993; Jacquemin et al. 1996). The Sd, as well as the TEA domain of its human homolog TEF-1, bound to the SRF-A, but not to the SRF-B fragment with high
affinity, comparable to that of both proteins binding to the Sph and
GT-IIC TEF-1 binding sites identified in the SV40 enhancer (Fig. 6B,C;
data not shown) (Davidson et al. 1988; data not
shown). Sd and TEF-1 TEA domains also bound specifically to the
vg quadrant MD2 element but with a lower affinity than to SRF-A (data not shown). Neither protein recognized a mutated GT-IIC site shown previously not to be bound by TEF-1 (Davidson et al. 1988).
The Sd and TEF-1 TEA domains recognized all sequences tested in a
similar manner, indicating that the two TEA domains have virtually
identical DNA-binding specificities (Fig. 6C). This is consistent with
the finding that TEF-1 can rescue sd mutant flies (Deshpande
et al. 1997). The pattern of mobility shifts of the Sd TEA domain bound
to the SRF-A fragment closely resembles the pattern of complexes
obtained by shifting the Sph fragment (Fig. 6C). The Sph fragment
contains two tandem TEF-1 binding sites that are bound cooperatively by
the TEF-1 TEA domain (Davidson et al. 1988). Inspection of the SRF-A
sequence revealed two tandem sets of potential binding sites that
resemble those in the Sph enhancer (A1 and A2; Fig. 6A,B).
|
To identify the specific sequences to which Sd bound, we performed DNase I footprinting with the Sd protein on the SRF-A and SRF-B regions (Fig. 6D). The Sd TEA domain protected one of the two tandem sets (A2), whereas neither the other set (A1) nor any other regions were protected, even at the highest protein concentration tested (Fig. 6D). As expected, SRF-B showed no footprint (not shown). To test whether the A2 binding sites were required for activation of SRF-A in tissue culture, we mutated these sites to sequences that were no longer bound specifically by Sd in gel mobility shift assays (Fig. 6C). When transfected into S2 cells, the activation of the mutant reporter plasmid by Vg and Sd was severely reduced (Fig. 7A). Similarly in Drosophila, although the SRF-A element was sufficient to drive expression specifically in the wing pouch (Fig. 7B), the mutated SRF-A lacking Sd binding sites was inactivated (Fig. 7C). Therefore, the sequence-specific binding of Sd to the SRF cis-regulatory element is required for enhancer activation.
|
Combinatorial control of wing-specific gene expression patterns by Vg/Sd and signaling inputs
Our results demonstrate that the activation of several genes in the
wing field requires Vg/Sd function. It is also known that for each of the cis-regulatory elements analyzed here, direct input(s) of particular signaling pathways are also required.
Specifically, the activation of the SRF intervein C element requires
both Vg/Sd and Hh signaling (U. Nussbaumer, J. Montagne,
J. Groppe, and M. Affolter, in prep.), the activation of the
vg boundary enhancer requires Vg/Sd and N
signaling (Kim et al. 1996), and the activation of the vg
quadrant enhancer requires Vg/Sd and Dpp signaling (Kim et al. 1996). Because these regulatory elements are not expressed in
all tissues in which the signals are active, nor in all wing cells in
which Vg/Sd are active, we deduce that neither the input of various signals nor of Vg/Sd alone are sufficient for
gene activation in vivo. Rather, our results suggest that the various wing-specific cis-regulatory elements require a combination of direct inputs, comprising the Vg/Sd selector function,
which restricts expression to the wing field, and at least one signal
transducer that mediates signaling inputs and hence, the pattern of
gene expression within the wing field. One prediction of this model is
that gene expression patterns within the wing field may be changed by
altering the signal-transducer binding sites within a
cis-response element. To test this, we changed the Suppressor of Hairless [Su(H)] binding site that mediates the N input in the
vg boundary enhancer (Kim et al. 1996) to sites for the
Cubitus interruptus (Ci) protein that transduces Hh signaling. This
switches the pattern of gene expression from a N-induced dorsoventral
stripe to a Hh-induced anteroposterior stripe while retaining the
restriction of gene activation to the wing disc (Fig. 8A).
|
| |
Discussion |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
Vg interacts with Sd to regulate wing-specific gene expression
Our genetic and biochemical results suggest that Vg regulates wing
identity by acting together with the Sd protein to control wing-specific patterns of gene expression. The requirement of Sd
function for Vg activity and nuclear localization in vivo, the synergy
of Vg and Sd in cotransfection experiments, the direct binding of Sd to
cis-regulatory sequences required for target gene activation,
and the demonstration of a physical interaction between Vg and Sd
(Simmonds et al. 1998) all support a model whereby the two proteins
form a transcriptional activator in which Sd is the sequence-specific
DNA binding component.
The vertebrate Sd homologs, the TEF proteins (for review, see Jacquemin
and Davidson 1997) also require specific coactivators for their
function (Xiao et al. 1991; Ishiji et al. 1992; Hwang et al. 1993), and
one of them was identified as the bHLH protein Max (Gupta et al. 1997).
Interestingly, these coactivators have a tissue specific distribution,
whereas the TEFs are widely expressed. Similarily, in
Drosophila, Sd is expressed and required in several imaginal
discs, whereas Vg functions specifically in the developing wing disc.
Thus, it is the spatial restriction of Vg expression that determines
the boundaries of the wing field.
Tissue-specific responses to signaling pathways: the role of selector genes
Our results demonstrate that the role of the Vg/Sd selector function is to directly regulate wing-specific cis-regulatory elements that also require particular signaling inputs. The patterns of gene expression induced in the wing disc are limited to cells in which both the selector genes and specific signaling pathways are active. The response of the SRF-A, vg boundary, and vg quadrant enhancers to Hh, N, and Dpp signaling are limited to the wing pouch by Vg/Sd and occur in different patterns because of their direct regulation by the Ci, Su(H), and Mad proteins, respectively (Fig. 8B). Furthermore, the finding that the changing of the Su(H) binding site into a Ci binding site in the vg boundary enhancer switches the pattern from a wing-specific dorsoventral N-regulated stripe to a wing-specific anteroposterior Hh-regulated stripe suggests that spatial expression patterns are determined by the sites for individual DNA-binding signal transducers.
One corollary of this model is that for any given signaling protein,
different selector proteins may be involved in directing tissue-specific responses in different organs and tissues. It has
recently been shown that tissue-specific enhancers in the embryo that
are regulated by Dpp also require the action of the Labial/Extradenticle (Grieder et al. 1997) or Tinman (Xu
et al. 1998) selector proteins to limit expression to the endoderm or mesoderm, respectively. We suggest that, in general, combinatorial control by selector proteins and common signal transducers at a
cis-regulatory level is required for the tissue- and
organ-specific responses of target genes to widely deployed signaling systems.
| |
Materials and methods |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
Ectopic expression of Vg and Sd
Ectopic expression of Vg was driven by the Gal4-UAS expression
system (Brand and Perrimon 1993). dpp(4A.3)-Gal4 (Morimura et
al. 1996) and Dll-Gal4 (Calleja et al. 1996) drivers were
used to activate UAS-Vg (Kim et al. 1996) in a specific spatiotemporal pattern. dpp-Gal4 expresses along the A/P
compartment boundary of leg and wing imaginal discs. Dll-Gal4
is expressed in the distal portions of leg and the wing pouch imaginal
tissues. Clones of ectopic Vg were generated by mating hs-FLPase;
UAS-Vg flies with flies carrying an actin
promoter-FRT-CD2-FRT-Gal4 transgene (Pignoni and Zipursky 1997).
Heat shock at 34°C for 30 min at 40-60 hr after egg laying induced
expression of FLPase catalyzing the site specific recombination between
the FRT sites, resulting in Gal4 expression in clones. Larvae were aged
2 days after heat shock and dissected at the late third instar stage.
The hs-sd transgene was induced by four heat shocks at 37°C for 45 min every 4 hr. Heat shocks were started at 70-80 hr after egg laying
and crawling third instar larvae were dissected for disc strainings.
Sd mutant clones
Mitotic recombination to generate sd mutant clones was
induced by 
-rays in progeny from a cross between
sd47m/FM7 flies and flies carrying a
heat-inducible 18M myc (Bailey and Posakony 1995). Larvae were
aged at 25°C an additional 48 hr after irradiation, heat-shocked for
2 hr at 36.5°C to induce Myc expression, and then allowed to recover
for 45-120 min at 25°C prior to dissection.
Immunohistochemistry
Antibody and X-gal stainings of imaginal discs were done as
described (Halder et al. 1998). Immunohistochemistry of transfected Drosophila S2 cells involved spotting cells onto a slide,
fixing in 4% paraformaldehyde followed by a 
20°C methanol wash,
blocking with 3% BSA, incubating with mouse anti-
-gal (Promega,
1:100) and rabbit anti-Vg (1:100) for 1 hr and then with
fluorescent secondary antibodies (Jackson Laboratories) for 1 hr. Topro
counterstaining was performed in the final washes. Antibodies to Sal
were provided by Reinhard Schuh (Max-Planck Institute für
Biophysikalische Chemie, Goettingen, Germany) and to SRF by Markus
Affolter (Biocenter, Basel, Switzerland).
Transient transfection of Drosophila S2 cells
vg and sd cDNAs were cloned into the pPacPL
expression vector (Koelle and Hogness 1992). Drosophila S2
cells were maintained in M3 medium with 12.5% heat-inactivated fetal
bovine serum (FBS) and 1% Pen-Strep (PS) at an average density of
6 × 106 cells/ml with the medium replaced
every 2 days. For transfection, cells were seeded at
2 × 106 the night before, washed, and resuspended in M3
without FBS and PS at 3.75 × 106/ml. A
mixture of lipofectin, DNA, and M3 was incubated 30 min at room
temperature before adding to S2 cells in M3. Following incubation for 6 hr at 25°C, M3 with FBS and PS was added to the cells, which were
then incubated for an additional 36 hr at 25°C. lacZ
reporter gene expression was assayed with chlorophenol
red--D-galactopyranoside as a substrate as described
(Rouet et al. 1992) except that cell lysis was achieved by use of a
hypotonic buffer (20 mM Tris, 10mM NaCl, 20%
glycerol, 0.2 mM EDTA, 0.1% NP-40) in place of quick freeze-thaws. This colorometic assay was quantified at 595 nm by a
standard 96-well plate reader. Relative activation values are
normalized to -Gal activity of the reporter gene alone in S2
cells. Reported activation of MD2, Vg-A, and Vg-B was with 0.1 µg
of Vg and 0.1 µg of Sd; SRF-A, SRF-A mutant, and SRF-B was with 0.1 µg of Vg and 0.03 µg of Sd expression plasmid. All transfections were performed in triplicate in at least three
independent experiments. Transfection efficiency ranged from 5% to
15% in independent experiments as assayed by antibody staining for Vg expression. The efficiency was not significantly different between reporter genes or in the presence/absence of Sd and was
consistent among triplicate samples. Representative data from a single
experiment is shown.
Reporter constructs
Reporter constructs were created by cloning subfragments of the
vg quadrant enhancer (Kim et al. 1996; MD2), the vg
boundary enhancer (Vg-A and Vg-B), and the SRF Intervein C enhancer
(SRF-A and SRF-B) (U. Nussbaumer, J. Montagne, J. Groppe, and M. Affolter, in prep.) into the hsp-lacZ-CaSpeR plasmid (Nelson
and Laughon 1993). In the mutant version of the SRF-A reporter
construct, the sequence of the A2 binding site was changed to
5'-AAAAATTAGTGGAATAAGT-3' (mutated bases underlined; the same mutations abolish binding of TEF-1
to high affinity sites in the GT-IIC and Sph enhancers (Davidson et al.
1988)). To replace the Su(H) site in the vg boundary enhancer,
the sequence ctagTGGGTGGTCgatccaaagTGGGTGGTCgtaccg, which contains
two tandem Ci-binding sites (shown in uppercase), was inserted into the
Su(H) site (Kim et al. 1996).
Purification of His-tagged TEA domains
Construction of the TEF-1 TEA domain expression plasmid was
described previously (Jacquemin et al. 1996). The Sd TEA domain expression plasmid was constructed by mutagenizing the codon that encodes the one amino acid that differs between the Sd and TEF-1 TEA
domains with PCR. Both plasmids express TEA domains tagged with six
histidines at their carboxyl terminus. Expression was induced for 2 hr
in BL21 Lys S cells and was purified on nickel chelate columns
(Novagen). Proteins were dialyzed against buffer containing 100 mM KCl, 10 mM Tris (pH 7.5), 250 µM
EDTA, 1 mM DTT, frozen, and stored at 80°C in binding
buffer (12% glycerol, 15 mM HEPES at pH 7.9, 50 mM
KCl, 4 mMMgCl2, 100 µg/ml BSA, 1 mM DTT). The TEA domains were nearly homogeneous as judged by SDS-PAGE.
Electrophoretic mobility-shift assays
Electrophoretic mobility shift assays were performed by incubating 1-3 fmoles of labeled template with 0.1-10 ng TEA domain and 5 ng of poly [d(AT)] in 25 µl of binding buffer. After 30 min at room temperature, complexes were separated on 6% polyacrylamide gels and standard 0.5× TBE buffer. Templates were annealed oligonucleotides that were radioactively labeled by fill in with Klenow on EcoRI overhangs on either side. Sequences of the upper strand oligonucleotides were Sph (5'-AATTCGCAGAAGTATGCAAAGCATGCATCTC-3'), GT-IIC (5'-AATTCAGCTGTGGAATGTGTGTC-3'), GT-IIC mutant (5'-AATTCACCTTTCTAATTTGTGTC-3'). SRF-A and SRF-B templates: The corresponding regions were PCR amplified with primers that contained EcoRI sites and subcloned into pCRII (Invitrogen). Fragments were then excised with EcoRI, gel purified, and radioactively labeled by fill-in reaction.
DNase I footprinting
A fragment spanning the SRF-A and SRF-B regions was PCR amplified
and subcloned into pCRII (Invitrogen). The insert was then cut on one
side with XhoI, radioactively labeled by fill-in reaction, and
gel purified after complete removal of the insert with
HindIII. A total of 30,000 cpm of this fragment was incubated
for 30 min together with the Sd TEA domain at room temperature in
incubation buffer containing 100 mM KCl, 15 mM
HEPES (pH 7.9), 12% glycerol, 4 mM MgCl2, 1 mM CaCl2, 1 mM DTT and 100 µg/ml BSA. For digestion, 5 µl of DNase solution
(1:10 dilution of Promega's RQ1 DNase I in binding buffer without
BSA) was added and the reaction stopped after 1 min by adding 140 µl of stop solution (final concentrations: 0.5% SD, 15 mM EDTA, 100 mM NaCl, 25 µg/ml
tRNA). After phenol/chloroform extraction and ethanol
precipitation, the samples were separated on a 6% polyacrylamide
sequencing gel. G+A sequencing reactions were done according to Maxam
and Gilbert (1980).
| |
Acknowledgments |
|---|
We thank John Bell, Ken Irvine, and Alfonso Martinez-Arias for communication of results prior to publication; Ute Nussbaumer, Jacques Montagne, and Markus Affolter for detailed information and valuable materials concerning the SRF enhancer; Ken Irvine for the UAS-sd stock; Irwin Davidson for provision of the TEF-1 expression construct; Scott Weatherbee for the image in Figure 1C; Mike Hoffmann for Drosophila S2 cells; Rosa Barrio, Reinhard Schuh, Markus Affolter, and William Chia for antibodies; Jim Williams for the hs-sd construct; Kathy Vaccaro for technical assistance and Jamie Wilson for help with manuscript preparation. G.H. holds a long-term fellowship from the European Molecular Biology Organization, P.P. is funded by a WARF fellowship, and MEK is a National Institutes of Health postdoctoral fellow. This work was supported by a grant from the National Science Foundation (A.S.L.) and by the Howard Hughes Medical Institute (S.B.C.).
The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked `advertisement' in accordance with 18 USC section 1734 solely to indicate this fact.
| |
Footnotes |
|---|
Received September 17, 1998; revised version accepted November 6, 1998.
4 These authors contributed equally to this work.
5 Corresponding author.
E-MAIL sbcarrol{at}facstaff.wisc.edu; FAX (608) 262-9343.
| |
References |
|---|
|
Top
Abstract
Introduction
Results
Discussion
Materials and methods
References
|
|---|
-Myosin heavy-chain gene expression.
Mol. Cel. Biol.
17:
3924-3936[Abstract].
CiteULike 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  A. Sawada, H. Kiyonari, K. Ukita, N. Nishioka, Y. Imuta, and H. Sasaki Redundant Roles of Tead1 and Tead2 in Notochord Development and the Regulation of Cell Proliferation and Survival Mol. Cell. Biol., May 15, 2008; 28(10): 3177 - 3189. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Zecca and G. Struhl Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation Development, August 15, 2007; 134(16): 3001 - 3010. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Zecca and G. Struhl Control of Drosophila wing growth by the vestigial quadrant enhancer Development, August 15, 2007; 134(16): 3011 - 3020. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Garg, A. Srivastava, M. M. Davis, S. L. O'Keefe, L. Chow, and J. B. Bell Antagonizing Scalloped With a Novel Vestigial Construct Reveals an Important Role for Scalloped in Drosophila melanogaster Leg, Eye and Optic Lobe Development Genetics, February 1, 2007; 175(2): 659 - 669. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Jacobsen, D. Cain, L. Paul, S. Justiniano, A. Alli, J. S. Mullins, C. P. Wang, J. P. Butchar, and A. Simcox Functional Analysis of Genes Differentially Expressed in the Drosophila Wing Disc: Role of Transcripts Enriched in the Wing Region Genetics, December 1, 2006; 174(4): 1973 - 1982. [Abstract] [Full Text] [PDF]
|
|
|
|
