1 The Salk Institute for Biological Studies, PO Box 85800, San Diego, CA 92186,
USA
2 Department of Biology, University of California, Riverside, CA 92521,
USA
* Present address: McGill University Centre for Research in Neuroscience, 1650
Cedar Avenue, Montreal, QC H3G 1A4, Canada
Present address: CyThera, San Diego, CA 92121, USA
Author for correspondence (e-mail:
jthomas{at}salk.edu)
Accepted 29 January 2003
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SUMMARY |
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Key words: LIM domain, Homeodomain, Drosophila, Wing development, Apterous, Chip
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INTRODUCTION |
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The developing wing of Drosophila has proven a tractable system in
which to study the function of complexes formed by LIM-HD proteins and their
co-factors (Fernandez-Funez et al.,
1998; Milan et al.,
1998
; Shoresh et al.,
1998
; Zeng et al.,
1998
; Milan and Cohen,
1999
; Rincon-Limas et al.,
2000
; Weihe et al.,
2001
). The wing imaginal disc is divided into distinct
lineage-restricted compartments along both the anteroposterior and
dorsoventral (DV) axes. In response to signaling via epidermal growth factor
receptor (Wang et al., 2000
;
Zecca and Struhl, 2002a
;
Zecca and Struhl, 2002b
), the
LIM-homeodomain protein Apterous (Ap) is expressed in the dorsal compartment
of the wing disc where it is required to establish an affinity boundary that
partitions the wing along the DV axis
(Cohen et al., 1992
;
Diaz-Benjumea and Cohen, 1993
;
Blair et al., 1994
). The DV
boundary of the wing disc differentiates into the wing margin, which lies at
the edge of the adult wing blade, and is decorated with mechanosensory and
chemosensory bristles distributed in a discrete DV pattern
(Palka, 1993
). Ap induces
Notch activation at the DV boundary through induction in dorsal cells of the
Notch ligand Serrate and the glycosyltransferase Fringe
(Irvine and Wieschaus, 1994
;
Kim et al., 1995
;
Panin et al., 1997
;
Klein and Arias, 1998
;
Micchelli and Blair, 1999
;
Rauskolb et al., 1999
;
Ju et al., 2000
;
O'Keefe and Thomas, 2001
).
This leads to the expression of the secreted morphogen Wingless in a stripe
that prefigures the margin, patterns the wing along the DV axis and directs
cell proliferation and wing outgrowth
(Diaz-Benjumea and Cohen,
1993
; Zecca et al.,
1996
; Neumann and Cohen,
1997
). Finally, through activation of the msh gene, Ap is
required to specify the dorsal identity of cells such as sensory bristles and
vein tissue (Milan et al.,
2001
).
The domains of Ldb1/Chip co-factors that are required for self-dimerization
and LIM interaction have been identified
(Jurata and Gill, 1997;
Breen et al., 1998
;
van Meyel et al., 1999
). In
Drosophila, it has been shown that Chip and Ap physically interact in
vivo to form a tetrameric complex comprised of two molecules of Ap bridged by
a Chip homodimer (Milan and Cohen,
1999
; van Meyel et al.,
1999
; van Meyel et al.,
2000
). This complex is required for Ap activity in DV patterning
and outgrowth of the wing and is subject to disruption by Drosophila
LMO (Bx FlyBase), a nuclear LIM-only protein that can compete with Ap
for binding to Chip and thereby modulate Ap activity
(Milan et al., 1998
;
Shoresh et al., 1998
;
Zeng et al., 1998
). In the
wing, LMO expression is upregulated by Ap, thus providing a mechanism for
negative feedback upon Chip/Ap tetrameric complexes and modulation of Ap
activity (Milan and Cohen,
1999
; Milan and Cohen,
2000
; Weihe et al.,
2001
).
Although Chip is required to dimerize and bring two molecules of Ap into a
tetrameric complex, we have hypothesized that it may also recruit other
proteins or co-factors required for correct transcriptional regulation of
target genes (van Meyel et al.,
2000). In the present study, we describe the identification and
characterization of members of the Ssdp family of proteins in mice and flies,
which specifically interact with Chip/Ldb proteins as shown here and by others
(Chen et al., 2002
). The N
terminus of Ssdp contains a recently described LUFS domain, which we find is
required for interaction with Chip. Chip binds Ssdp through a highly conserved
domain that is distinct from domains for LIM binding and homodimerization, and
Chip is required for correct nuclear localization of Ssdp. In vivo, we find
that Ssdp is capable of modifying Chip function in wing development. Although
null mutations of ssdp are cell-lethal in clones of cells in the
developing wing disc, clones mutant for a hypomorphic allele of ssdp
give rise to margin, outgrowth and cell identity defects that are strikingly
similar to those produced by mutations of Chip and ap.
Intriguingly, proteins with structural similarity to Ssdp and Chip have
recently been shown to cooperate with one another to regulate the expression
of a homeotic gene functioning during development of plants
(Conner and Liu, 2000
;
Franks et al., 2002
). These
results suggest that molecular interactions of the kind between Ssdp and
Chip/Ldb proteins are evolutionarily ancient and may supply a fundamental
function in the regulated control of transcription.
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MATERIALS AND METHODS |
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Yeast qualitative interaction assays
Plasmids were transformed into yeast strain Y187, plated, and colonies were
assayed for ß-galactosidase activity by the filter lift method as
described in the Clontech Matchmaker system protocol. A positive result was
scored if blue color developed upon incubation for 3 hours at 30°C. All
DBD-Ldb1 constructs and DBD-Ssdp2(1-50) were cloned into pAS2-1,
DBD-Ssdp2(1-100) and DBD-Ssdp(1-98) from Drosophila were in pGBT9.
All AD-Ssdp2 constructs were in pGAD10, and AD-Ldb1 and AD-Chip constructs
were in pACT2. The DBD control vector pLAM5'-1 encodes human Lamin C.
Negative controls were assayed for each construct to ensure that
auto-activation or nonspecific binding did not occur: DBD-Lamin C was tested
with most AD constructs, except Chip vectors where empty DBD vector pAS2-1 was
used; empty AD vector pACT2 was tested with DBD constructs.
Immunoprecipitation
In vitro transcription and translation were performed according to the
manufacturers instructions (TNT Reticulocyte Lysate System, Promega) with or
without 35S-methionine (NEN Life Sciences products). Ten µl of
each protein was mixed and 20 µl of binding/wash buffer (50 mM HEPES, pH
7.5; 250 mM NaCl; 0.1% NP-40; 200 µM ZnCl2 and MgCl2)
was added. After incubation for 2 hours on ice, reactions were cleared with
protein A sepharose, and immunoprecipitated with anti-Flag M2 agarose beads
(Kodak/IBI). Eluted samples were analyzed on 4-12% acrylamide gradient gels
(NuPAGE, Invitrogen), and the results observed using autoradiography.
Fly strains and genetics
The strains EP(3)3004, EP(3)3097 and l(3)neo48 were obtained from the
Berkeley Drosophila Genome Project collection
(Cooley et al., 1988;
Rorth et al., 1998
). Recently,
the strain KG03600 (Roseman et al.,
1995
) has been identified as an insert in the ssdp locus,
but was used in only some of the analyses presented here. The
ssdpL7 and ssdpL5 alleles were the
result of imprecise excisions generated by mobilization of the EP(3)3097
P-element using
2,3 as a source of transposase
(Tsubota and Schedl, 1986
;
Robertson et al., 1988
). Each
of these was fully lethal in complementation tests with
ssdpl(3)neo48. A number of precise excisions that fully
complemented ssdpl(3)neo48 were recovered, indicating that
lethality in EP(3)3097 was due to insertion of this P-element in the
ssdp locus. DNA sequencing of the breakpoints of the
ssdpL7 deficiency revealed that it is a complete null
allele of ssdp resulting from the deletion of 3363 base pairs (bp) of
DNA from the insertion site of the EP(3)3097 P-element through the entire
coding region of ssdp plus 941 bp of sequence downstream of the 340
bp 3' untranslated region (UTR). This breakpoint lies 2002 bp away from
the nearest predicted gene (CG14313), which is of unknown function. Southern
analysis strongly suggests that ssdpL5 results from the
deletion of
1750 bp of coding sequence, but the boundaries of this
deficiency were not determined by DNA sequencing. In all analyses where it has
been examined, ssdpL5 has had effects identical to those
of ssdpL7, suggesting that it too is a null allele. All
crosses and embryo/larval collections were performed at 25°C, unless
stated otherwise.
Balanced stocks for each of the ssdp mutations were maintained over TM3 marked by actin-lacZ. This dominant marker was used to score the timing of lethality for various mutant allelic combinations. Homozygous mutants were assessed for viability at the first and third instar larval stages, and upon eclosion of adults.
DNA constructs for transgenic Drosophila
We obtained two Drosophila ssdp cDNAs (LD23161 and LD37723) and
found each to contain the entire open reading frame of the ssdp gene
(Research Genetics). Using LD37723 as a template for polymerase chain
reactions (PCR), SsdpFL and Ssdp2-92 constructs were created by a
previously described strategy to include five C-terminal Myc epitopes and two
stop codons (van Meyel et al.,
1999
). In a similar fashion, Chip
387-426 was created from a
Chip cDNA (Morcillo et al.,
1997
). Each of these was fully sequenced, then cloned into pUASt
(Brand and Perrimon, 1993
). UAS
lines were generated by germline transformation
(Rubin and Spradling, 1982
)
and, for each construct, a minimum of 20 independent lines were created and
tested for expression. Those lines that exhibited the strongest, GAL4-directed
Myc expression were selected for analysis.
In situ hybridization and immunostaining
In situ hybridization was performed on Drosophila embryos in whole
mount, and on dissected wandering third instar larvae. A 1.4 kb digoxigenin
(DIG)-labeled antisense cRNA probe was synthesized using SP6 RNA polymerase
and StuI-cut LD37723 ssdp cDNA. For immunofluorescence
staining, we drove Myc-tagged UAS transgene expression in muscles 21-24 with
apGAL4 (Calleja et al.,
1996), and crossed this combination into the
Chipe5.5 mutant background
(Morcillo et al., 1997
). We
stained dissected embryos with mouse anti-Myc (9E10) at a dilution of 1:50,
and secondary antibodies conjugated to Cy3 (Jackson ImmunoResearch) at 1:500.
Confocal analysis was performed on a Zeiss confocal station and imaged with
the LSM510 software (Zeiss). Images were compiled with Adobe Photoshop
6.0.
Mosaic analysis
Individuals carrying chromosomes recombinant for ssdp mutations
and FRT inserts at 82B were selected on media containing Geneticin
(Invitrogen) and subsequently tested by complementation for viability against
mutant alleles of ssdp. Timed embryo collections were subjected to
heat-shock (1 hour, 38°C) at discrete stages of larval development either
36 hours, 48 hours, 72 hours or 96 hours after egg-laying (AEL). After
eclosion, individuals of the genotypes listed below were analyzed for the
presence of clones as indicated by the cell-autonomous marker pwn
(Heitzler et al., 1996), which
is seen as pin-shaped hairs (trichomes) with spurs on each mutant cell, and
truncated bristles. For microscopic examination, wings were removed, immersed
in isopropanol followed by methyl salicylate, then mounted on glass slides in
Canada Balsam.
ssdp mutants
hsFLP38pwn/pwn;FRT,
ssdpL7/FRT,Dp pwn+
hsFLP38pwn/pwn;FRT, ssdpL5/FRT,Dp pwn+
hsFLP38pwn/pwn;FRT, ssdpl(3)neo48/FRT,Dp pwn+
Control
hsFLP38pwn/pwn;FRT, P(w+)90E/ FRT,Dp
pwn+
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RESULTS |
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To verify the specific interaction between Ldb and Ssdp, we assayed for co-immunoprecipitation of proteins translated in rabbit reticulocyte lysates (Fig. 1). We co-incubated various combinations of Ldb and Ssdp proteins labeled with 35S-methionine and/or tagged with the FLAG epitope. Co-incubation was followed by immunoprecipitation with anti-FLAG antibody-conjugated agarose beads and analysis by SDS-PAGE. We found Ssdp2 protein to be efficiently immunoprecipitated by Ldb1 (lane 2), but not by the LIM-HD protein Lhx3 (lane 4). As expected, Lhx3 was capable of binding Ldb1 (lane 1) and importantly, was able to immunoprecipitate Ssdp2 in the presence of Ldb1 (lane 3), arguing for the formation of a ternary complex in which Lhx3 and Ssdp2 are each bound to Ldb1. Additional control experiments showed that the Ldb1-Ssdp2 complex was not immunoprecipitated in the absence of the FLAG epitope (lane 5), and only a small amount of either Ssdp2 or Ldb1 binds nonspecifically to the beads (lanes 5 and 6).
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To map the interaction with Ssdp2 more precisely, two internal deletions of
10 amino acids each were constructed, Ldb1235-244 and
Ldb1
214-223 (Fig. 2D).
Ldb1
235-244 binds to Ssdp but Ldb1
214-223 does not. As a
positive control, the LIM domains of Lhx3 were shown to bind both of these
mutants (Ldb1
214-223 shown in Fig.
2D).
Both Ssdp2 and Ldb1 have orthologous counterparts in Drosophila, called Ssdp and Chip. As shown in Fig. 2E, fly Ssdp residues 1-98 can bind strongly to Chip, and this interaction is dependent upon amino acids 387-426 of Chip. Chip residues 387-435 are 94% identical to Ldb1 amino acids 201-249, and we have named this region the Ldb1/Chip conserved domain (LCCD). Taken together, the results indicate that the N terminus of Ssdp proteins bind Ldb/Chip proteins in a region that is distinct from the two domains needed to form the tetrameric complex, namely the dimerization domain (DD) and the LIM interaction domain (LID).
The interaction domains of Ssdp and of Ldb1/Chip have been highly
conserved through evolution
Searches of the NCBI databases indicate that Ssdp proteins comprise a
family of highly related proteins in which there are four members in humans
(Castro et al., 2002), three in
mice and only one in Drosophila. Comparison of primary sequence from
Ssdp proteins from these and other species reveals a high degree of amino acid
identity, particularly within the first 100 amino acids. A schematic of the
overall protein structure comparing mouse Ssdp2 and fly Ssdp is shown in
Fig. 3A, and a sequence
alignment of the N-terminal sequences for several family members is shown in
Fig. 3B. Between flies and mice
there is 90% identity over this N-terminal region. As is the case for all
family members, the remainders of these proteins are characterized by an
unusually high proportion of proline, glycine and methionine residues. For
example, of the 352 amino acids of Drosophila Ssdp from amino acids
93-445, 21% are proline, 27% are glycine and 9% are methionine, for a total of
57% of all residues. Within this overall architecture, there are three small
regions that are highly conserved across species
(Fig. 3A).
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Like the LUFS domain of Ssdp proteins, the LCCD of Ldb1/Chip has been highly conserved through evolution, with 94% identity between mice and flies over a stretch of 49 amino acids (Fig. 3C).
Nuclear localization of Drosophila Ssdp is dependent upon the LUFS
domain and Chip
The single ssdp gene in flies has been annotated CG7187 by the
Berkeley Drosophila Genome Project and a number of corresponding cDNAs have
been isolated. The gene consists of two exons, the second of which contains
the single open reading frame which encodes a 445 residue polypeptide. We
designed epitope-tagged versions of Drosophila Ssdp in which five
copies of the Myc epitope were fused to the C terminus of full-length Ssdp
(SsdpFL) or a mutant lacking amino acids 2-92 (Ssdp2-92). These
constructs were used to generate transgenic lines in which transgene
expression was under the control of UAS sequences
(Brand and Perrimon, 1993
). We
used different GAL4 driver lines to express these recombinant proteins in a
variety of cell types, including neurons and muscles. SsdpFL localized to
nuclei, with no staining in the cytoplasm
(Fig. 3D). By contrast,
Ssdp
2-92 was found throughout the cytoplasm
(Fig. 3E). Therefore nuclear
localization of Ssdp is dependent upon the Chip-interacting LUFS domain,
despite the fact that this region does not appear to encode a nuclear
localization sequence (NLS). To address whether Chip, which does have an NLS,
is required for translocation of Ssdp into the nucleus, we tested whether
SsdpFL is properly localized to the nucleus in Chipe5.5
null mutants. In contrast to wild-type, SsdpFL was distributed throughout the
cytoplasm of cells lacking zygotic Chip
(Fig. 3F). Occasionally, we
could detect staining in nuclei in addition to cytoplasmic staining. This may
reflect residual activity in these embryos of maternally provided Chip. These
results argue that nuclear targeting of Ssdp occurs through a Chip-dependent
mechanism.
Drosophila ssdp is expressed in neural and imaginal
tissues
The pattern of ssdp expression was determined using in situ
hybridization of digoxigenin-labeled antisense cRNA probes to embryos and
third instar larvae. In embryos of syncytial blastoderm stage, ssdp
transcript was ubiquitous, suggesting that there is maternal contribution. By
the time of germband extension, although still widespread, expression appears
to be enriched in the developing central nervous system (CNS)
(Fig. 4A). During germband
retraction this enrichment of transcript in the embryonic CNS is more apparent
(Fig. 4A), such that by stage
13-14 ssdp expression is largely restricted to the brain and ventral
nerve cord (Fig. 4B). Closer
examination of the pattern of expression in the ventral nerve cord suggests
that expression occurs in all neurons of the CNS, with no major subclasses
excluded (Fig. 4C). This
pattern of expression is maintained through later stages of embryogenesis (not
shown). In third instar larvae, ssdp is no longer detected in the
ventral nerve cord (Fig. 4D),
but moderate ssdp expression is observed in the optic lobes of the
brain hemispheres (Fig. 4D,E).
High levels of ssdp expression are observed in imaginal discs,
including the anterior region of the antennal-eye disc
(Fig. 4E), the wing and haltere
discs (Fig. 4F) and all leg
discs (not shown), as well as in the salivary gland (not shown). With the
exception of the eye-antennal disc, expression in imaginal discs is largely
uniform.
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Ssdp can modify Ap/Chip complex activity in the wing
Ap is expressed in the dorsal compartment of the wing disc and is required
to establish the DV affinity boundary, the wing margin, wing outgrowth and
dorsal-specific wing structures such as sensory bristles
(Diaz-Benjumea and Cohen,
1993; Blair et al.,
1994
). In the absence of Ap, the wing fails to develop
(Cohen et al., 1992
). We and
others have previously shown that Ap functions through a tetrameric complex in
which two molecules of Ap are bridged by a homodimer of Chip
(Milan and Cohen, 1999
;
van Meyel et al., 1999
).
Chip mutants interact genetically with ap to cause
disruptions of the wing margin (Morcillo
et al., 1997
), and clones of Chip mutant cells in the
wing disc behave like ap mutant clones
(Fernandez-Funez et al., 1998
;
Milan and Cohen, 2000
),
causing ectopic wing margins and outgrowths.
In contrast to a previous study (Chen et
al., 2002), we detected no phenotypes in simple trans-heterozygous
combinations of a null allele of Chip with any ssdp alleles
used here, including ssdpKG03600 and the two null alleles
ssdpL5 and ssdpL7. Nor did we detect
any phenotypes in transheterozygous combinations of ap and
ssdp. Thus, to address the role of Ssdp in the function of
Chip/LIM-HD complexes in vivo, we used the GAL4-UAS system to reduce Ap/Chip
complex activity to levels that would be sensitive to the effects of reducing
ssdp gene dosage. We used apGAL4, a GAL4
P-element insertion in the ap gene, which faithfully expresses GAL4
in Ap-expressing cells (Calleja et al.,
1996
), to drive expression of UAS transgenes in the dorsal
compartment of the wing disc.
Over-expression of UAS-Chip has been shown previously to disrupt
wing patterning by titrating endogenous Ap into incomplete complexes in which
LID domains of Chip molecules remain vacant
(Fernandez-Funez et al., 1998;
Milan and Cohen, 1999
;
van Meyel et al., 1999
).
Relative to controls (Fig. 6A),
such wings are small and lack regular structure, and the wing margin is poorly
demarcated (Fig. 6B). These
phenotypes resemble hypomorphic ap mutants, and can be completely
suppressed by simultaneous overexpression of UAS-ap
(Fernandez-Funez et al., 1998
;
Milan and Cohen, 1999
;
van Meyel et al., 1999
). This
indicates that the stoichiometry between Ap and Chip is an important factor in
the formation of functional complexes. We examined the effect of removing one
copy of the ssdp gene and found that the resulting flies had little
or no residual wing tissue, consistent with a further reduction of the
activity of the complex (Fig.
6C).
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Finally, we compared the effects of Chip overexpression with those produced
by expression of a Chip variant lacking the LCCD (ChipLCCD).
Chip
LCCD is capable of self-dimerization and binding to Ap, but it
cannot bind Ssdp. If Ssdp were required for function of the complex,
Chip
LCCD would be predicted to have a more potent dominant-negative
effect on the function of the complex than would Chip itself, as the latter
can still recruit Ssdp. Expression of Chip
LCCD with
apGAL4 consistently produced more extreme wing defects
than Chip (Fig. 6F).
Chip
LCCD sequesters Ap into nonfunctional complexes, but it cannot bind
Ssdp. Therefore, removal of one copy of ssdp would not be expected to
suppress the phenotype caused by Chip
LCCD, and indeed it does not (data
not shown). Collectively, these results argue that in addition to forming the
dimeric bridge for two molecules of Ap, Chip also recruits Ssdp to the
complex.
Generation of ssdp mutant clones in the wing disc gives rise
to defects that resemble closely those of ap and Chip
Clones of ap mutant cells in the dorsal compartment of the wing
disc induce an ectopic wing margin and therefore ectopic wing outgrowth. These
ap mutant cells differentiate ventral wing margin structures, despite
the fact that they remain in the dorsal compartment. Chip mutant
clones induced in the dorsal compartment give rise to strikingly similar
phenotypes (Fernandez-Funez et al.,
1998; Milan and Cohen,
2000
). The effects of Chip clones are influenced both by
the timing of their induction as well as their position within the disc
(Milan and Cohen, 2000
). For
example, clones induced later (third instar) resulted in ectopic margin
tissue, but did not lead to outgrowth.
If Ssdp were an additional member of the Ap/Chip complex, then mutations of ssdp would be predicted to give rise to mutant phenotypes similar to those of ap and Chip. To test this, we used the FRT/FLP recombinase system to induce clones of cells mutant for ssdp in an otherwise heterozygous animal. Clones were generated in larvae at second and third instar by heat-shock induction at 36 hours, 48 hours, 72 hours or 96 hours after egg laying (AEL). The effects of clone induction were observed in newly eclosed adults. Clones of mutant cells were identified by the presence of the cell-autonomous marker pawn (pwn). Each of the mutant alleles ssdpL7, ssdpL5 and ssdpl(3)neo48 were tested, as was a control chromosome with no mutation, and the experiment was repeated on four separate occasions, each time observing many individuals of each genotype.
In controls, many clones of various sizes were induced, as evidenced by the presence of pwn mutant cells (Fig. 7A). These clones occurred on both the ventral and dorsal surfaces of the wing blade, but no mutant phenotypes were ever observed. By contrast, clones of cells mutant for either ssdpL7 or ssdpL5 (as marked by pwn) were never observed on either surface of the wing blade, indicating that both alleles have cell-lethal effects in the wing disc. In addition, there were fewer than the expected number of adults eclosing of the appropriate genotype for clone induction, suggesting that the cell-lethal effects, presumably in tissues other than the wing, lead to decreased viability.
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Induction of ectopic margin bristles was the most commonly observed effect of dorsal ssdp mutant clones. They were primarily observed in proximity to a clone near the native anterior wing margin and comprised at least one row of extra sensory bristles (Fig. 7E,F). Most ectopic bristles were not marked by pwn, indicating they were induced by the neighboring mutant (pwn) cells. ssdpl(3)neo48 mutant clones that occurred within the margin, rather than near it, resulted in the loss of dorsal-specific sensory bristles (Fig. 7G). Occasionally a large clone was observed to straddle the dorsoventral boundary, and in these instances, the entire margin, including some nearby non-margin tissue, was lost (Fig. 7H).
In general, there was a striking resemblance between the phenotypes resulting from ssdpl(3)neo48 mutant clones and those reported for clones of Chip or ap. This provides strong evidence that Ssdp is an important additional component of Chip/Ap transcriptional complexes in vivo.
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DISCUSSION |
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In mice, Ssdp1 and Ssdp2 are expressed broadly (A.D.A, unpublished). Knockout mice for Ssdp2 have been generated and preliminary evidence suggests they die early during embryogenesis (A.D.A., S. Pfaff and S.-K. Lee, unpublished), making it difficult to assess the role of Ssdp2 in LIM-HD functions. Using Drosophila as a model, we have shown that mutations in ssdp can modify the activities of Chip and the LIM-HD protein Ap in vivo, and that the wing phenotypes caused by ssdp mutant clones are strikingly similar to those produced by mutations of Chip and ap. Our findings provide strong evidence that Ssdp is a functional component of Chip/Ap complexes during development.
The N termini of Ssdp proteins contain a recently described LUFS domain, which we find is sufficient for interaction with Chip. Within Chip, the highly conserved LCCD is required for Ssdp binding and is a domain that is distinct from those for LIM interactions and homodimerization. It is therefore possible that Chip/Ap tetrameric complexes also include two molecules of Ssdp, each bound specifically to one of the two Chip molecules in the complex.
Ssdp requires the LUFS domain and Chip for correct localization to the
nucleus; in the absence of either, Ssdp remains cytoplasmic. Taken together,
these results suggest that Ssdp and Chip bind to one another in the cytoplasm,
whereupon Ssdp is brought to the nucleus to participate with Chip and Ap in
transcriptional regulatory complexes. SSDP was first identified as a DNA
binding protein in avian cultured cells, notable because it bound in a
sequence-specific manner to a poly-pyrimidine sequence in the promoter region
of the 2(I) collagen gene. We do not know whether the ability of Ssdp
to bind DNA is required to support the function of the Chip/Ap tetrameric
complex in vivo, and as yet the DNA-binding domain of Ssdp is
uncharacterized.
Given that Chip/Ldb proteins bind the LIM domains of all LIM-HD proteins,
we think it is likely that Ssdp also participates in the function of other
LIM-HD proteins in the imaginal tissues and nervous system where it is
expressed. However, it is also likely that Ssdp has additional functions
outside the context of LIM-HD proteins. The mild cleft observed in the dorsal
thorax of adult ssdp hypomorphs is similar to that of mutants of the
GATA factor pannier. Pannier has been shown to complex with Chip and
basic helix-loop-helix proteins and promote development of the dorsal thorax
(Ramain et al., 2000). It is
possible that Ssdp too may play a role in the activity of this complex
following recruitment by Chip.
Furthermore, our finding that clones mutant for null alleles of ssdp are cell lethal in the wing disc, whereas Chip and ap clones are not, indicates that Ssdp proteins must have additional functions in wing tissue that are independent of either Chip or Ap.
The LUFS domain is a novel protein interface for transcription
regulation in plants and animals
The domains that mediate the interaction between Ssdp proteins and Chip/Ldb
are highly conserved, even in plants where the Arabidopsis LUFS
domain-containing protein LEUNIG cooperates with SEUSS, a protein that shares
similarity with Chip/Ldb proteins. Like Ssdp and Chip/Ldb, LEUNIG and SEUSS
interact in a yeast two-hybrid assay, although the domains responsible for
this interaction have not been mapped
(Franks et al., 2002). In
addition, genetic analyses has revealed that these proteins cooperate in the
transcriptional regulation of AGAMOUS, a homeotic gene functioning in flower
development. However, domains within LEUNIG outside of the LUFS domain are
different from Ssdp proteins and include glutamate-rich regions and WD40
repeats (Conner and Liu,
2000
). LEUNIG is probably a transcriptional co-repressor, based on
its regulation of AGAMOUS plus its overall structural similarity to the yeast
co-repressor Tup1 (Liu and Meyerowitz,
1995
; Conner and Liu,
2000
). Given that the effects of ssdp mutation in the
Drosophila wing phenocopy those of Chip and ap, we view Ssdp
as a likely activator of the complex, not a repressor, and propose that this
fundamental difference between LEUNIG and Ssdp proteins lies in the functional
domains C-terminal to the LUFS domain where these proteins bear no resemblance
to one another.
The intriguing conservation from plants to vertebrates of the interaction between the LUFS domain and sequences within Chip/Ldb and SEUSS proteins suggest a fundamentally important interaction to enable regulated control of transcription. However, unlike Ldb1 and Chip, SEUSS has no LIM interaction domain, nor are there any LIM-HD proteins in plants. It is possible that interactions between LUFS domains and Chip/Ldb/SEUSS proteins exemplify an ancient transcriptional regulatory function that has been recruited by LIM-HD proteins in animals by the addition of a LIM interaction domain to Chip/Ldb.
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ACKNOWLEDGMENTS |
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