From the School of Biological Sciences, University of
East Anglia, Norwich, Norfolk NR4 7TJ, United Kingdom and the
¶ Division of Cell and Developmental Biology, Wellcome Trust
Biocentre, University of Dundee,
Dundee DD1 5EH, Scotland, United Kingdom
Received for publication, September 5, 2002, and in revised form, December 12, 2002
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ABSTRACT |
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Members of the spalt family of zinc
finger-containing proteins have been implicated in development and
disease. However, very little is known about the molecular function of
spalt proteins. We have used biochemical approaches to characterize
functional domains of two chick spalt homologs, csal1 and
csal3. We show that csal1 and csal3 proteins repress
transcription and that they can interact with each other. Furthermore,
we found that truncated chick spalt proteins, similar to the truncated
spalt protein expressed in the human congenital disorder Townes-Brocks
syndrome, affect the nuclear localization of full-length spalt. Our
findings have implications for the understanding of Townes-Brocks
syndrome and the role of spalt genes in normal development.
We propose that truncated spalt can exert a dominant negative effect
and is able to interfere with the correct function of full-length
protein, by causing its displacement from the nucleus. This could
affect the transcriptional repressor activity of spalt and DNA binding. Spalt protein truncations could also affect the function of other spalt
family members in various tissues.
This work focuses on the functional analysis of two chick homologs
of the spalt family, csal1 and
csal3.1 As
described previously csal1 is most closely related to human Hsall1 and mouse msall1, whereas csal3
is most closely related to Hsall3 and msall3
(1-7). Spalt proteins are important in a number of developmental
processes, cell fate decisions, and organogenesis. Spalt
(sal) was first isolated in Drosophila and
encodes a protein characterized by multiple double zinc finger motifs
of the C2H2 type, commonly found in
transcription factors (8). Expression of sal is found in
various tissues in Drosophila embryos and larvae (8). Early
in development sal acts as a region-specific homeotic gene
and is required for the specification of head and tail regions (9). At
later stages sal regulates pattern formation and cell fate
decisions in the wing disc, the trachea, and in sensory organs of the
peripheral nervous system (10-14). In addition it was shown recently
that sal is regulated by the homeobox gene
ultrabithorax (ubx), which can directly repress
multiple cis-elements in the sal promoter (15).
Vertebrate sal homologs have been identified in mouse,
Xenopus, and Medaka (1, 5, 7, 16-18), where they
are expressed in various tissues including central nervous system,
heart, pronephros, and limb/fin buds. In human, three homologs of
sal have been isolated (3, 19). Mutations in one of these
genes, termed Hsall1 or SALL1, which result in a
premature truncation and loss of the C2H2 zinc
finger motifs, cause Townes-Brocks syndrome
(TBS),2 an autosomal dominant
disorder. TBS is characterized by anorectal, ear, and hand
malformations, in particular preaxial polydactyly (2, 20). These
observations show that vertebrate sal homologs play
important roles in the development of a number of organ systems. In
addition, mice deficient in msall1, a homolog of
Hsall1, die perinatally from severe kidney failure caused by
incomplete ureteric bud outgrowth. However, neither heterozygous nor
homozygous mice mimic the TBS phenotype, and it was suggested that
msall1 deficiency may be compensated for by
msall2 and/or msall3 in mice. Alternatively, the
mutant Townes-Brocks protein may exert a dominant negative effect and
eliminate the function of all spalt proteins in humans (21).
The structure of spalt proteins suggested that they may act as
transcriptional regulators. This was supported by the finding that a
highly related Drosophila protein spalt-related (salr), can
bind to DNA in vitro (22). More recently it was shown that fusion proteins of mouse sall1 and human SALL1 with the GAL4 DNA binding domain suppress transcription of a GAL4 reporter plasmid. This
repression required the amino-terminal single zinc finger, which, in
case of msall1, can bind to histone deacetylase (23, 24).
We have used in vitro biochemical approaches to elucidate
further the molecular function of spalt proteins. We demonstrate here
that chick spalt proteins, csal1 and csal3, repress transcription from
a reporter plasmid. The proteins physically interact with each other in
cultured cells, and the conserved glutamine-rich region located within
the amino-terminal third of spalt proteins is necessary for this
interaction. We also demonstrate that "TBS-like" truncated chick
proteins can still interact with wild-type protein and with each other.
We show that truncated csal1 protein is localized to the cytoplasm of
cells, whereas the full-length protein is exclusively nuclear. In
addition, in the presence of truncated spalt proteins, nuclear
localization of csal1 is abrogated. Together these findings provide new
evidence for the molecular mechanism underlying TBS.
Probes and in Situ Hybridization--
Digoxigenin-UTP-csal1 and
FITC-UTP-csal3 antisense RNA probes were generated, and in
situ hybridization was carried out as described previously (4, 6).
Probes were detected separately, and anti-digoxigenin antibody coupled
to alkaline phosphatase (Roche Molecular Biochemicals) was developed
with 5-bromo-4-chloro-3-indolyl-phosphate (Roche) at 350 µg/ml.
Embryos were then washed extensively, fixed in 4% paraformaldehyde,
heat treated at 65 °C, and anti-FITC antibody coupled to alkaline
phosphatase (Roche) was detected with Magenta Phos (Sigma) at 175 µg/ml.
Plasmid Constructs--
To generate an in-frame fusion with the
GAL4 DNA binding domain the open reading frames of csal1 was
subcloned into p1012GAL4 using HindIII and
XbaI, and csal3 was subcloned using
EcoRI and XhoI. The cloning vector, p1012GAL4 and
the reporter plasmids, pG5TKCAT and pG0TKCAT, were a gift from Dr. Neil
Perkins, University of Dundee (25).
To construct HA- and FLAG-tagged spalt proteins we performed PCR
mutagenesis and replaced the internal stop codon with an XhoI site for cloning into pCMV1-HA or pCMV1-FLAG (26).
Truncations and deletions were generated using site-directed
mutagenesis to introduce internal XhoI sites by PCR into the
tagged spalts. PCR products were digested with
DpnI (Roche) and transformed into competent DH5 Cell Transfections and CAT Assays--
HEK293 cells were
transfected with 12.5 µg of the reporter plasmid and 2.5 µg of the
GAL4 fusion construct by calcium phosphate and CAT assays carried out
as described (27, 28). Briefly, protein extract of transfected cells
was prepared in lysis buffer (0.25 M Tris, pH 7.8) by
repeated freeze-thaw cycles, protein concentration was determined using
the Bradford method (Bio-Rad), and equal amounts of protein (10 µg)
were incubated with [14C]chloramphenicol and acetyl-CoA
for 20 min at 37 °C. Reaction products were separated on a silica
TLC plate with 90% chloroform and 10% methanol; after drying the
plates conversion of [14C]chloramphenicol to
[acetyl-14C]chloramphenicol was quantitated
using a Molecular Dynamics PhosphorImager.
Immunoprecipitation--
HEK293 cells were transfected with 10 µg of each plasmid (27). After 48 h protein was extracted with
Nonidet P-40 lysis buffer (50 mM Tris, pH 8, 150 mM NaCl, 1% Igepal) in the presence of complete proteinase
inhibitor (Roche) by incubating on ice for 20 min and centrifugation at
13,000 rpm for 5 min (29). Protein extracts were precleared with 15 µl of protein A-agarose (Sigma) for 20 min at 4 °C and then
immunoprecipitated with 10 µl of either anti-HA.11 (Babco) or anti
FLAG M2 (Sigma) and shaken for 1 h at 4 °C. After this, 30 µl
of protein A-agarose was added and shaken for 1 h at 4 °C. The
protein A-agarose pellet was washed four times in 1 ml of Nonidet P-40
lysis buffer and boiled in 20 µl of Laemmli buffer before SDS-PAGE
separation and Western blotting. Western blots were performed with the
same antibodies and detected with either a secondary anti-mouse
IgG-horseradish peroxidase (Jackson) or protein A-horseradish
peroxidase (Sigma) using the enhanced chemiluminescence detection
reagent (Amersham Biosciences).
Immunofluorescence and Microscopy--
COS-7 cells were grown on
coverslips and transfected with 5 µg of each plasmid by calcium
phosphate (27). After 48 h cells were fixed in 4%
paraformaldehyde, permeabilized in 0.1% Triton X-100, and proteins
were detected with primary antibodies, anti-HA.11 (Babco) or anti-FLAG
polyclonal (Sigma), and anti mouse-FITC and anti rabbit-TRITC secondary
antibodies (Jackson). Immunostaining was analyzed using a Zeiss
Axioplan2 and a Leica TCS SP2 confocal microscope.
Coexpression of csal1 and csal3 in Limb Development--
We have
shown previously that csal1 and csal3 are
expressed during limb development (4, 6). In the present study we have
used double in situ hybridization to determine the extent to
which csal1 and csal3 transcripts might be
coexpressed. Both chick spalt genes were detected in
developing wing and leg buds from Hamburger-Hamilton (HH) (30) stages
23-25 (Fig. 1, A-D). At
HH23, csal1 and csal3 transcripts were both
expressed in the distal most region of limb buds with a slightly
broader expression of csal1 (Fig. 1A).
Subsequently, csal3 expression became more highly
restricted, and both transcripts overlapped in posterior limb bud
mesenchyme (Fig. 1, B and C). At HH25,
csal3 expression began to decrease, and faint purple
staining was identified in a patch of mesenchyme cells (Fig.
1D, white line tracing). Transcripts of both
genes were also detected in other areas of the embryo, in particular
the developing neural tube and the tail bud (Fig. 1C). This
raises the possibility that csal1 and csal3 could
interact functionally during development.
GAL4 Fusions of Csal1 and Csal3 Mediate Transcriptional
Repression--
It was demonstrated recently that fusion proteins of
both human and mouse sall1 with the GAL4 DNA binding domain (GAL4DBD) can repress transcription from a GAL4 responsive reporter plasmid (23,
24). To assess whether chick spalt proteins can also function as
transcriptional repressors, we performed similar experiments and fused
the GAL4DBD to the amino-terminal end of chick csal1 and csal3. These
plasmid constructs were cotransfected into HEK293 cells along with a
reporter construct, G5TKCAT, which contains five GAL4 binding sites in
front of the HSVTK promoter driving CAT gene expression (25). CAT
activity was quantitated and compared with the CAT activity obtained
with the plasmid vector containing only the GAL4DBD, which was set to
be 100%. This showed that, similar to human and mouse sall1,
csal1-GAL4DBD and csal3-GAL4DBD fusion proteins can repress
transcription, 4-fold and 6.5-fold, respectively (Fig.
2, left two columns).
To test whether transcriptional repression depends on DNA binding we
cotransfected GAL4 spalt fusion proteins with a reporter plasmid
lacking GAL4 binding sites (G0TKCAT; Fig. 2, right two
columns). In this situation we observed an ~2-fold reduction in
transcription activity.
Physical Interactions of Csal1 and Csal3--
We next wanted to
investigate whether csal1 and csal3 can interact to form higher order
structures. We generated HA- and FLAG-tagged versions of both chick
spalt proteins (Fig. 3). HEK293 cells
were cotransfected with these constructs, and immunoprecipitations were
performed using protein extracts followed by Western blot analysis. As
a control, protein extracts from cells transfected with either csal1-HA
or csal3-FLAG were immunoprecipitated with both anti-HA and anti-FLAG
antibodies. In neither case did we observe nonspecific
cross-reactivity, and protein was detected on a Western blot only after
immunoprecipitation with the appropriate antibody (Fig.
4A, lanes 3 and
6). We found that immunoprecipitation of csal1-FLAG with an
anti-FLAG antibody resulted in coprecipitation of csal1-HA-tagged
protein, demonstrating a physical interaction between multiple csal1
proteins (Fig. 4B, lane 6). Similarly, immunoprecipitation of csal3-FLAG with an anti-FLAG antibody resulted in coprecipitation of csal3-HA-tagged protein, suggesting that csal3
forms homo-oligomers (Fig. 4C, lane 6). Because
csal1 and csal3 are coexpressed in limb
development we next examined whether they could interact with each
other. Using protein extracts of cells transfected with csal1-HA and
csal3-FLAG expression constructs we could detect csal1-HA protein after
immunoprecipitation of csal3-FLAG, demonstrating that csal1 and csal3
can interact in HEK293 cells (Fig. 4D, lane
6).
The Glutamine-rich Domain of Spalt Mediates Protein
Interactions--
To identify domains in csal1 and csal3 which can
mediate spalt protein interactions, we generated truncated versions of
the proteins and tested their ability to interact in
coimmunprecipitation assays (Fig. 3, A and B).
Initially we produced HA-tagged versions of csal1 containing amino
acids 1-806 and 1-290. Both of these truncations retain the
amino-terminal single zinc finger and the glutamine-rich region but
lack some or all of the C2H2 double zinc finger
motifs. Mutations in Hsall1 which cause TBS result in the
loss of all C2H2 double zinc finger motifs (2).
Immunoprecipitation of csal1-1-806-HA with an anti-HA antibody
resulted in coprecipitation of full-length csal1-FLAG (Fig.
5A, lane 6), and
immunoprecipitation of csal1-1-290-HA with an anti-HA antibody
resulted in coprecipitation of full-length csal1-FLAG, demonstrating
that the first 290 amino acids are sufficient to mediate interactions
with full-length csal1 (Fig. 5A, lane 5).
Similarly, immunoprecipitation of csal3-HA with an anti-HA antibody
resulted in coprecipitation of the truncated csal3-1-282-FLAG protein,
indicating that amino acids 1-282 of csal3 are sufficient to interact
with full-length csal3 (Fig. 5B, lane 4). Next we
investigated whether csal1-1-290 and csal3-1-282 can interact with
each other. We found that immunoprecipitation of csal1-1-290-HA
resulted in coprecipitation of csal1-1-290-FLAG and csal3-1-282,
respectively (Fig. 5C, lanes 5 and 6).
Together these experiments demonstrate that TBS-like amino-terminal
truncations of csal1 and csal3 are sufficient to mediate interactions
with full-length proteins and with each other.
We generated further deletions of both csal1 and csal3 to identify the
domain within the amino terminus which mediates physical interactions
between chick spalt proteins. HA-tagged csal1 truncations were
generated, encompassing amino acids 1-65, 1-218, 1-240, and FLAG-tagged csal3 truncations were generated encompassing amino acids
1-85, 1-230, and 1-252. As depicted in the schematic representation csal1-1-65 and csal3-1-85 contain only the amino-terminal single zinc
finger motif, csal1-1-218 and csal3-1-230 contain in addition the
intervening region up to the glutamine-rich domain, and csal1-1-240 and csal3-1-252 terminate immediately after the glutamine-rich domain.
We also generated a FLAG-tagged csal3 construct with an internal
deletion of the glutamine-rich domain, termed csal3- Subcellular Localization of Csal1 and Csal3--
We examined the
subcellular localization of the two spalt proteins in COS-7 cells,
transfected with tagged versions of both chick spalt proteins. Using
anti-HA or anti-FLAG antibodies and fluorescence microscopy, we
detected csal1-HA exclusively in the nucleus (Fig.
7, A, C, and
D), whereas csal3-FLAG was found exclusively in the
cytoplasm (Fig. 7, F-H). Confocal microscopy confirmed that
csal3-FLAG was excluded from the nucleus (data not shown). Interestingly, when csal1-HA and csal3-FLAG were cotransfected we
observed a clear change in the subcellular localization of csal1-HA. In
the presence of csal3, csal1 was detected in the cytoplasm colocalizing
with csal3-FLAG (Fig. 7, I, J, and L). In the absence of csal3, we have been unable to detect cytoplasmic expression of csal1, indicating that subcellular localization of csal1
is altered by csal3. This suggests that csal3 may change the function
of csal1 in regions of the embryo where both spalt proteins are
expressed.
Expression of Truncated Spalt Proteins Affects the Localization of
Csal1--
We coexpressed full-length csal1-HA with csal1-1-290-FLAG
to determine what effect this might have on protein distribution. In
contrast to full-length csal1, truncated csal1 protein (csal1-1-290) was no longer restricted to the nucleus but instead could be detected throughout the cell (Fig. 7, M-P). When this
truncated form of csal1 was cotransfected with full-length csal1
protein we detected full-length csal1 in the cytoplasm with apparently
increased levels immediately around the nucleus (Fig. 7,
Q-T, compare Q and A). In contrast,
the csal1 truncation that lacks the glutamine-rich region
(csal1-1-218) was no longer able to relocalize full-length csal1
protein from the nucleus to the cytoplasm (Fig. 7, U-X, compare U, Q, and A). These
experiments indicate that TBS-like amino-terminal truncations of spalt
proteins alter the normal localization of full-length csal1, and this
effect is mediated by the glutamine-rich region. To confirm that the
glutamine-rich region is required for relocalization, we cotransfected
csal1-HA and csal3- The experiments presented here demonstrate for the first time that
spalt proteins can interact with each other. The conserved glutamine-rich domain is necessary to mediate protein interactions of
csal1 and csal3, two chick spalt family members. Furthermore, we
determined the subcellular distribution of tagged csal1 and csal3
proteins and found that the localization of full-length csal1 is
altered in the presence of csal3 or truncated forms of csal1.
Relocalization required the presence of the conserved glutamine-rich region. We discuss our findings and their implications for spalt function during normal embryogenesis and in TBS.
Structural Motifs of Spalt Proteins--
The structure of spalt
proteins, in particular the presence of multiple zinc finger motifs of
the C2H2 type, suggests a role as a
transcription factor. This is supported by the finding that Drosophila spalt-related protein can bind DNA and by the
recent evidence demonstrating the ability of human and mouse sall1 to repress transcription from a heterologous reporter (23, 24). Transcriptional repressor activity of msall1 is mediated by the amino-terminal zinc finger, which was shown to recruit histone deacetylase (24). This is in contrast to human sall1, where transcriptional repression was observed with GAL4DBD fusions, but this
was independent of histone deacetylase (23). The amino-terminal zinc
finger domain is highly conserved in all vertebrate members of the
spalt family described so far, suggesting that transcriptional repression may be a common property of different spalts from different species. Consistent with this, we found that both csal1 and csal3 repress transcription. However, our results suggest that only part of
the sal-mediated transcriptional repression activity depends on direct
binding to the active promoter. In addition, there may be a
contribution that is independent of DNA binding. The mechanisms underlying this phenomenon need further investigation, ideally in the
context of a native target promoter of sal proteins, which has yet to
be identified.
We found that csal3 is expressed in the cytoplasm rather than in the
nucleus of transfected COS-7 cells. This seems to rule out a role as a
transcriptional repressor in vivo. However, it is
conceivable that stimulation of cells with an appropriate growth factor
could cause translocation of csal3 into the nucleus where it could act
on DNA. Alternatively, the role of csal3 could be to act as a negative
regulator of csal1 function. In cells coexpressing both csal1 and csal3
we observed that csal1 is cytoplasmic and the removal of csal1 from the
nucleus could provide a mechanism for abrogating its functions. These
explanations are not exclusive, and csal3 could regulate csal1 by its
effect on subcellular localization and also have a role as a
transcriptional repressor in its own right.
The glutamine-rich region is highly conserved in all invertebrate and
vertebrate spalt family members isolated so far, but its function has
never previously been characterized. Our experiments demonstrate that
this domain is required for interaction of csal1 and csal3 with
themselves and with other spalt proteins. The glutamine-rich region is
sufficient to mediate spalt interactions, when present within the amino
terminus. Furthermore, in the absence of this domain, we do not observe
any interactions between the spalts, demonstrating that this domain is
necessary. It is unclear at present whether glutamine-rich motifs of
two spalt molecules bind directly to each other or to another motif
within the amino terminus or whether they mediate binding to an
unrelated protein, which will then in turn interact with another spalt molecule.
Role of Spalt Proteins in Development--
We showed previously
that the onset of csal1 expression at HH17 in wing and leg
bud precedes that of csal3. During subsequent stages of limb
bud outgrowth up to HH25, csal1 expression is restricted to
the distal third of wing and leg bud and the apical ectodermal ridge.
After HH26, csal1 expression was no longer detected in limb buds. In
contrast, csal3 is only transiently expressed in HH18 wing
buds, expression can be detected again from HH23-HH26 in a region of
posterior mesenchyme (4, 6). Here we showed csal1 and
csal3 transcripts overlap in a distinct region of the developing limb buds (Fig. 1), and full-length csal1 and csal3 proteins
interact in coimmunoprecipitation experiments (Fig. 4D). These observations raise the distinct possibility that csal proteins may also form homo- and heterodimers in vivo and interact
functionally during embryogenesis to pattern the developing limb.
We showed here that csal1 and csal3 show a distinct subcellular
distribution with csal1 being mainly nuclear and csal3 mostly found in
the cytoplasm. Although this localization to separate compartments of
the cell seems to preclude a physical interaction in vivo we
found that the distribution of csal1 changes in presence of csal3 (Fig.
7). Thus it is interesting to speculate that in embryonic development
csal3 protein may act as a modifier of csal1 by altering csal1
localization and consequently affecting its function in the nucleus.
Both chick spalt genes are coexpressed in a subset of limb mesenchyme
cells, and limb development is affected in patients with TBS.
Implications for Townes-Brocks Syndrome--
Expression of a
truncated Hsall1 protein in the human congenital syndrome TBS has
indicated an important role for spalt in normal development. To date it
is unclear whether the complex phenotype in TBS is caused by
haploinsufficiency or a dominant negative effect of the truncated
protein. Interestingly, mouse embryos with homozygous deletions of
csal1 have apparently normal limbs and show no sign of the polydactyly
and other phenotypes that are typically seen in TBS patients. Instead
they display a severe kidney phenotpye. The heterozygous mice appear
normal (21), and this finding argues against haploinsufficiency as the
underlying mechanism. Instead, the TBS gene product may act in a
dominant negative manner. In this scenario, it would not be surprising
that complete loss of msall1 function does not mimic the TBS phenotype
because other members of the family may be able to compensate.
There is growing evidence that the Townes-Brocks protein retains
important functional domains (23, 24, and this report), and this is
more consistent with the possibility that the TBS phenotype is caused
by dominant negative interference with the normal function of other
proteins. The truncated Hsall1 proteins expressed in TBS patients are
equivalent to csal1-1-290 and csal3-1-298. All proteins include the
most amino-terminal single zinc finger as well as the glutamine-rich
region, whereas they have lost all other zinc finger motifs (2, Fig.
3). Our experiments show that csal1-1-290 and csal3-1-298 can
still interact with full-length and truncated forms of csal1 and csal3
in coprecipitation assays (Figs. 5 and 6). In addition, both
truncations localize to the cytoplasm and affect the nuclear
localization of full-length csal1 in COS-7 cells (Fig. 7). Thus our
experiments strongly suggest that the mutant Hsall1 proteins expressed
in TBS patients may retain the ability to interact with other members
of the spalt family. Therefore, the phenotype may in part be the result
of a functional interference of truncated Hsall1 protein with different spalt proteins in different tissues.
There are other possible mechanisms by which a dominant
negative effect might be created. As described in this paper, truncated csal1 has the ability to relocate full-length csal1. Second, truncated spalt may sequester corepressors, such as histone deacetylase, or it
may form inactive complexes unable to bind DNA. All of these could have
a role in TBS, and further work is required to establish which of these
mechanisms are operating.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Escherichia coli using standard protocols. Plasmids were
digested with XhoI and the vector religated to generate
tagged truncations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
csal1 and csal3
transcripts are coexpressed in limb development. Double
in situ hybridization detecting csal1
(light blue) and csal3 transcripts
(purple) in limb buds of HH23 (A), HH24
(B and C), and HH25 (D) chick embryos.
At HH23 (A), the purple staining within the area
of light blue staining identifies the region where both
transcripts are expressed. In D, the white line
tracing and the asterisk (*) mark the purple
staining that starts to fade because csal3 expression begins
to decrease at this stage. A, dorsal view of HH23 wing buds;
B, dorsal and C, lateral views of HH24 leg buds;
D, lateral view of HH25 embryo; nt, neural tube;
tb, tail bud.
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Fig. 2.
Csal1 and csal3 repress transcription from a
reporter plasmid. CAT activity was measured in protein extracts of
cells transfected with 12.5 µg of either the G5TKCAT or G0TKCAT
reporter plasmids and 2.5 µg of the vector, p1012GAL4, containing
only the GAL4DBD. The CAT activity obtained in the presence of vector
alone was set at 100%, and CAT activity in experimental samples was
plotted relative to that as -fold reduction. After transfection with
2.5 µg of p1012GAL4-csal1, encoding a csal1 GAL4DBD fusion protein,
or transfection with 2.5 µg of p1012GAL4-csal3, encoding a csal3
GAL4DBD fusion protein, we observed a 4-fold or 6.5-fold reduction in
CAT activity with the G5TKCAT reporter (first two bars). In
contrast, both fusion proteins caused a 2.3-fold reduction in CAT
activity with the G0TKCAT reporter (third and fourth
bars). The results shown are representative of three independent
experiments.
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Fig. 3.
Schematic representation of tagged protein
constructs used and alignment of the glutamine-rich region in human and
chick. A, csal1 proteins; B, csal3
proteins. White ovals, amino-terminal zinc finger of the
C2HC type; dark gray ovals, glutamine-rich
domain; light gray ovals, zinc finger motifs of
the C2H2 type. C, alignment of 22 amino acids, encompassing the conserved glutamine-rich region of
Hsall1, csal1, and csal3. This region is identical in Hsall1 and csal1.
The amino acids that are different in csal3 are shaded in
gray; see also Ref. 6.
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Fig. 4.
Chick spalt proteins interact in HEK293
cells. HEK293 cells were transfected with plasmids expressing
tagged forms of csal1 and csal3 as indicated by (+) above each
lane in each panel. Protein extracts were
prepared, and immunoprecipitation (IP) was followed by
Western blotting. Antibodies used for immunoprecipitation are indicated
above each lane, and antibodies used for Western blotting
are indicated on the left of each panel;
T, total protein. A shows transfections of HEK293
cells with either csal1-HA (lanes 1, 3, and
5) or csal3-FLAG (FL; lanes 2,
4, and 6). Lanes 1 and 2,
detection of csal1-HA and csal3-FLAG in total protein lysates;
lane 3, detection of csal1-HA precipitated with anti-HA
antibody; lane 6, detection of csal3-FLAG precipitated with
anti-FLAG antibody. In B-D, lanes 1,
3, and 5 are extracts from mock transfected
HEK293 control cells. B, cotransfection of csal1-HA and
csal1-FLAG; lane 2, detection of csal1-HA and csal1-FLAG in
total protein extracts; lane 4, detection of csal1-HA
protein precipitated with anti-HA antibody; lane 6,
detection of csal1-HA protein coprecipitated with csal1-FLAG using the
anti-FLAG antibody. C, cotransfection of csal3-HA and
csal3-FLAG; lane 2, detection of csal3-HA and csal3-FLAG in
total protein extracts; lane 4, detection of csal3-HA
protein precipitated with anti-HA antibody; lane 6,
detection of csal3-HA protein coprecipitated with csal3-FLAG using the
anti-FLAG antibody. D, cotransfection of csal1-HA and
csal3-FLAG; lane 2, detection of csal1-HA and csal3-FLAG in
total protein extracts; lane 4, detection of csal1-HA
protein precipitated with anti-HA antibody; lane 6,
detection of csal1-HA protein coprecipitated with csal3-FLAG using the
anti-FLAG antibody.
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Fig. 5.
The amino-terminal regions of chick spalts
mediate protein interaction. HEK293 cells were transfected with
plasmids expressing tagged forms of csal1 and csal3 as indicated by a
(+) above each lane in each panel. Protein
extracts were prepared, and immunoprecipitation (IP) was
followed by Western blotting. Antibodies used for immunoprecipitation
are indicated above each lane, and antibodies used for
Western blots are indicated on the left of each
panel. In A and C, lanes 1,
4, and 7 are extracts from mock transfected
HEK293 control cells. In B, lanes 1,
3, and 5 are extracts from mock transfected
cells. A, cotransfection of csal1-HA truncations (1-290 and
1-806) with csal1-FLAG (FL); lanes 2 and
3, detection of csal1-HA truncations and csal1-FLAG in
total protein (T) extracts; lanes 5 and
6, detection of csal1-FLAG protein coprecipitated with
csal1-1-290-HA and csal1-1-806-HA using anti-HA antibody; lanes
8 and 9, detection of csal1-FLAG protein precipitated
with the anti-FLAG antibody. B, cotransfection of csal3-HA
and csal3-1-282-FLAG; lane 2, detection of csal3-HA and
csal3-1-282-FLAG in total protein extracts; lane 4,
detection of csal-1-282-FLAG protein coprecipitated with csal3-HA and
the anti-HA antibody; lane 6, detection of csal-1-282-FLAG
protein precipitated with the anti-FLAG antibody. C,
cotransfection of csal1-1-290-HA with csal1-1-290-FLAG or
csal3-1-282-FLAG; lanes 2 and 3, detection of
csal1-1-290-HA, csal1-1-290-FLAG, and csal3-1-282-FLAG in total
protein extracts; lane 5, detection of csal1-1-290-FLAG
protein coprecipitated with csal1-1-290-HA and the anti-HA antibody;
lane 6, detection of csal3-1-282-FLAG protein
coprecipitated with csal1-1-290-HA and the anti-HA antibody;
lane 8, detection of csal1-1-290-FLAG protein precipitated
with the anti-FLAG antibody; lane 9, detection of
csal3-1-282-FLAG protein precipitated with the anti-FLAG
antibody.
Q-FLAG (Fig.
3B). Immunoprecipitations followed by Western blot analysis demonstrated that only csal1-1-240 and csal3-1-252 proteins were able
to interact with csal1 and csal3 proteins. Immunoprecipitation of
csal1-1-240-HA resulted in coprecipitation of csal1-1-290-FLAG (Fig.
6A, lane 6). In
contrast, immunoprecipitation of either csal1-1-218-HA or
csal1-1-65-HA did not result in coprecipitation of csal1-1-290-FLAG
(Fig. 6A, lanes 7 and 8,
respectively). Furthermore, immunoprecipitation of csal1-1-240-HA
resulted in coprecipitation of csal3-1-282-FLAG (Fig. 6B,
lane 6). As expected, immunoprecipitation of csal1-1-218-HA
and csal1-1-65-HA did not result in coprecipitation of
csal3-1-282-FLAG (Fig. 6B, lanes 7 and
8). In similar experiments we found that only csal3-1-252
was able to interact with other spalt proteins (Fig. 6, C
and D). Immunoprecipitation of csal3-HA resulted in
coprecipitation of csal3-1-252-FLAG (Fig. 6C, lane 6). In contrast, immunoprecipitation of csal3-HA did not result in
coprecipitation of either csal3-1-230-FLAG or csal3-1-85-FLAG (Fig.
6C, lanes 7 and 8, respectively). The
bands migrating at a slightly higher molecular mass to csal3-1-252
observed in Fig. 6, C and D, lanes
5-12, are caused by detection of IgG light chains with protein A
and are also seen in mock transfected controls (Fig. 6, C
and D, lanes 5 and 9).
Immunoprecipitation of csal1-1-290-HA resulted in coprecipitation of
csal3-1-252-FLAG (Fig. 6D, lane 6) but not
csal3-1-230-FLAG or csal3-1-85-FLAG (Fig. 6D, lanes 7 and 8, respectively). Finally,
immunoprecipitation of csal1-HA does not result in coprecipitation of
csal3 protein, which lacks the 22 amino acids encompassing the
glutamine-rich region (csal3-
Q-FLAG, Fig. 6E, lane
6). These experiments show that the conserved glutamine-rich domain in csal1 and csal3 is necessary for interaction with other spalt
proteins.
View larger version (30K):
[in a new window]
Fig. 6.
The glutamine-rich region of chick spalt
proteins is necessary for protein interactions. HEK293 cells were
transfected with plasmids expressing tagged forms of csal1 and csal3 as
indicated by (+) above each lane in each panel.
Protein extracts were prepared, and immunoprecipitation (IP)
was followed by Western blotting. Antibodies used for
immunoprecipitation are indicated above each lane, and
antibodies used for Western blotting are indicated on the
left of each panel; T, total protein.
In A-D, lanes 1, 5, and 9 are extracts from mock transfected HEK293 control cells, and
lanes 2-4 show the detection of tagged proteins in total
protein extracts. A, cotransfection of csal1-1-290-FLAG
(FL) with HA-tagged deletion constructs of csal1;
lanes 10-12, detection of csal1-1-290-FLAG protein
precipitated with anti-FLAG antibody; lane 6, detection of
csal1-1-290-FLAG protein coprecipitated with csal1-1-240-HA using the
anti-HA antibody. B, cotransfection of csal3-1-282-FLAG and
HA-tagged csal1 deletion constructs; lanes 10-12, detection
of csal3-1-282-FLAG protein precipitated with anti-FLAG antibody;
lane 6, detection of csal3-1-282-FLAG protein
coprecipitated with csal1-1-240-HA using the anti-HA antibody.
C, cotransfection of csal3-HA and FLAG-tagged
csal3 deletion constructs; lanes 10-12, detection of
FLAG-tagged csal3 truncated proteins precipitated with anti-FLAG
antibody; lane 6, detection of csal3-1-252-FLAG protein
coprecipitated with csal3-HA using the anti-HA antibody. D,
cotransfection of csal1-1-290-HA and FLAG-tagged csal3 deletion
constructs; lanes 10-12, detection of FLAG-tagged csal3
truncated proteins precipitated with anti-FLAG antibody; lane
6, detection of csal3-1-252-FLAG protein coprecipitated with
csal1-1-290-HA using the anti-HA antibody. E shows protein
extracts of mock transfected cells (lanes 1, 4,
and 7), cotransfection of csal1-HA and csal3-FLAG
(lanes 2, 5, and 8), and
cotransfection of csal1-HA and csal3- Q-FLAG (lanes 3,
6, and 9); lane 5, detection of
csal3-FLAG coprecipitated with csal-HA. Compare with lane 6 with no coprecipitation of csal3-
Q-FLAG and csal1-HA.
View larger version (46K):
[in a new window]
Fig. 7.
Subcellular localization of csal1 is affected
by csal3 and by truncated csal1 proteins. Immunofluorescence of
COS-7 cells transfected with tagged proteins, csal1-HA
(A-D), csal3-FLAG (E-H), csal1-HA and
csal3-FLAG (I-L), csal1-1-290-FLAG (M-P),
csal-HA and csal1-1-290-FLAG (Q-T), csal1-HA and
csal1-1-218-FLAG (U-X), csal1- HA and csal3- Q-FLAG
(FL) (Y-B'). Cells were stained with an
FITC-coupled anti-HA antibody (A, E,
I, M, Q, U, and
Y), or a TRITC-coupled anti-FLAG antibody (B,
F, J, N, R, V,
and Z), or with 4,6-diamidino-2-phenylindole (C,
G, K, O, S, W,
and A'). Merged images are shown in D,
H, L, P, T, X,
and B'. The areas in which HA-tagged and FLAG-tagged
proteins overlap appear in yellow. In all transfections
equal amounts of each plasmid were used (5 µg).
Q-FLAG. Expression of csal3-
Q-FLAG was
observed throughout the cell, including expression in the nucleus.
However, expression of csal3-
Q-FLAG did not result in a change of
csal1-HA localization, which remained exclusively nuclear (Fig. 7,
Y-B', compare B' and L).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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ACKNOWLEDGEMENTS |
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We thank Dr. Mette Mogensen and David Moss for help with microscopy and image capture, Dr. Grant Wheeler for stimulating discussions, and Dr. Neil Perkins for the CAT reporter plasmids, G5TKCAT and G0TKCAT, and the GAL4 fusion vector, p1012GAL4.
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FOOTNOTES |
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* This work was supported in part by a Wellcome Trust Research Career Development Fellowship and a Wellcome Trust project grant (to A. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a Medical Research Council studentship.
To whom correspondence should be addressed: School of
Biological Sciences, University of East Anglia, Earlham Rd., Norwich, Norfolk NR4 7TJ, U. K. E-mail: a.munsterberg@uea.ac.uk.
Published, JBC Papers in Press, December 13, 2002, DOI 10.1074/jbc.M209066200
1 The second chick homolog, which we previously named csal2, will now be called csal3 because it is most closely related to human Hsall3 (3, 6). In this article we have used the acronym "H" to indicate human and "m" to indicate mouse before the acronym sall for spalt-like. The nomenclature committee for the human and mouse genome consider SALL for the human and Sall for the mouse genes as sufficient.
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ABBREVIATIONS |
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The abbreviations used are: TBS, Townes-Brocks syndrome; CAT, chloramphenicol acetyltransferase; CMV, cytomegalovirus; FITC, fluorescein isothiocyanate; GAL4DBD, GAL4 DNA binding domain; HA, hemagglutinin; HEK, human embryonic kidney; HH, Hamburger and Hamilton; HSV, herpes simplex virus; TK, thymidine kinase; TRITC, tetramethylrhodamine isothiocyanate.
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