Skirball Institute of Biomolecular Medicine and Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA
* Author for correspondence (e-mail: treisman{at}saturn.med.nyu.edu)
Accepted 15 May 2003
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SUMMARY |
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Key words: TRAP, ARC, Transcription, Adhesion, Compartment, Boundary, Drosophila
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INTRODUCTION |
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The posterior compartment of the wing disc expresses the selector gene
engrailed (en), which encodes a homeodomain transcription
factor (Tabata et al., 1995).
The secreted protein Hedgehog (Hh) is also limited to the posterior
compartment, but can only signal to cells in the anterior compartment, where
the Hh-responsive transcription factor Cubitus interruptus (Ci) is present
(Schwartz et al., 1995
;
Tabata and Kornberg, 1994
).
Cells just anterior to the anterior-posterior (AP) compartment boundary
respond to Hh by expressing Decapentaplegic (Dpp), a long-range organizer of
pattern throughout the wing disc (Basler
and Struhl, 1994
; Lecuit et
al., 1996
; Nellen et al.,
1996
; Tabata and Kornberg,
1994
). In addition, Hh signaling alters the affinity of these
cells, preventing them from mixing with cells that do not receive the Hh
signal or are unable to respond to it
(Blair and Ralston, 1997
;
Rodriguez and Basler, 1997
).
En is thought to make an additional contribution to cell affinity by
regulating unknown target genes in posterior cells
(Dahmann and Basler,
2000
).
An analogous system operates along the dorsal-ventral axis of the wing
disc. The LIM domain protein Apterous (Ap) acts as a selector for the dorsal
compartment (Diaz-Benjumea and Cohen,
1993). By activating dorsal-specific expression of the Notch (N)
ligand Serrate and the glycosyltransferase Fringe, which makes N
preferentially sensitive to the ventrally expressed ligand Delta (Dl), Ap
allows N activation specifically at the dorsal-ventral (DV) boundary
(Bruckner et al., 2000
;
Doherty et al., 1996
;
Kim et al., 1995
;
Panin et al., 1997
). N then
activates a stripe of Wingless (Wg), a concentration-dependent organizer of
the wing pouch (Diaz-Benjumea and Cohen,
1995
; Neumann and Cohen,
1997
; Rulifson and Blair,
1995
; Zecca et al.,
1996
). Ap also controls the expression of genes that regulate cell
affinity, preventing mixing between cells of the dorsal and ventral
compartments (Blair et al.,
1994
; Milan and Cohen,
1999
). Two of its target genes that may contribute to this process
are capricious (caps) and tartan (trn);
both encode leucine-rich repeat proteins that are specifically expressed in
the dorsal compartment at the time when affinity differences along this
dimension are established (Milan et al.,
2001
). Misexpression of either caps or trn in
the ventral compartment leads to cell death or to movement towards the DV
boundary; however, dorsal cells still maintain their dorsal affinity in the
absence of both genes (Milan et al.,
2001
). In addition to ap activity, N signaling is
required to prevent cells from crossing the boundary in either direction
(Micchelli and Blair, 1999
;
Milan and Cohen, 2003
;
O'Keefe and Thomas, 2001
;
Rauskolb et al., 1999
).
Sequence-specific transcription factors require the assistance of cofactors
to recruit RNA polymerase II and the basal transcriptional machinery. One
widely used co-activator is the mediator complex, which was first described in
yeast and has now been isolated from human and mouse cells and from
Drosophila (Malik and Roeder,
2000; Rachez and Freedman,
2001
). Many transcriptional activators, as well as some
repressors, require the mediator complex in order to regulate transcription in
vitro, even on naked DNA templates (Boyer
et al., 1999
; Fondell et al.,
1996
; Gu et al.,
1999
; Ito et al.,
1999
; Malik et al.,
2000
; Naar et al.,
1999
; Rachez et al.,
1999
; Ryu et al.,
1999
). The largest mediator-related complexes that have been
isolated contain about 20 subunits, but they seem to be divisible into
functional submodules. Smaller complexes, called positive cofactor 2 (PC2) or
the cofactor required for Sp1 activation (CRSP), are sufficient for
co-activator activity with a number of activators in vitro
(Malik et al., 2000
;
Ryu et al., 1999
) and directly
interact with the C-terminal domain of RNA polymerase II
(Naar et al., 2002
),
suggesting that they represent a core complex containing the essential
activator functions. In addition to the essential subunits of this complex,
some subunits may act as adaptors for specific transcription factors. The
TRAP220 subunit appears to be an adaptor for nuclear receptors; the
ligand-binding domains of these receptors bind specifically to TRAP220 in
vitro in a ligand-dependent manner (Ge et
al., 2002
; Hittelman et al.,
1999
; Kang et al.,
2002
; Yuan et al.,
1998
). Similarly, the Xenopus ARC105 subunit shows
specific interactions with Smad2 and Smad3, transcription factors in the Nodal
signaling pathway (Kato et al.,
2002
), and the mouse SUR2 (ABCC9 Mouse Genome Informatics)
subunit is specifically required for the activity of E1A-CR3, and of ELK1 that
has been phosphorylated by ERK (Stevens et
al., 2002
).
Two of the subunits absent from the core complex to which a function has
not yet been assigned by in vitro studies are the largest proteins in the
complex, TRAP240 and TRAP230. The presence of these two subunits, and of Cdk8
and Cyclin C, and the absence of CRSP70, differentiate ARC, a large complex,
from CRSP. ARC and CRSP have distinct activities in vitro
(Taatjes et al., 2002).
Distant homologs of these four proteins in yeast, Srb8-11, also form an
accessory subcomplex that has been implicated in transcriptional repression
(Boube et al., 2002
;
Lee et al., 2000
;
Song and Carlson, 1998
;
Song et al., 1996
).
We isolated mutations in the skuld [skd; previously named
blind spot (Gutierrez et al.,
2003)] and kohtalo (kto) genes, which encode
Drosophila homologs of TRAP240 and TRAP230, respectively, based on
their identical loss-of-function phenotypes in the eye disc
(Treisman, 2001
). Unlike
dTrap80 and dMed6, components of the core PC2/CRSP complex, Skd and Kto are
not required for cell proliferation or survival
(Boube et al., 2000
;
Gim et al., 2001
;
Treisman, 2001
). Here, we
examine the effects of skd and kto mutations on patterning
of the wing discs. We show that both genes again have identical functions, and
that they regulate the differences in cell affinity that create compartment
boundaries. We provide both genetic and biochemical evidence supporting the
model that these two proteins act in concert, probably as a submodule of the
mediator complex.
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MATERIALS AND METHODS |
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Antibodies and immunohistochemistry
Eye and wing discs were stained as described
(Lee and Treisman, 2001).
Antibodies used were rat anti-Ci (Motzny
and Holmgren, 1995
), rabbit anti-ß-galactosidase (Cappel,
1:5000), mouse anti-Wg (Ng et al.,
1996
), rat anti-Elav (Robinow
and White, 1991
), rabbit anti-Atonal
(Jarman et al., 1993
), rabbit
anti-Dephrin (Bossing and Brand,
2002
) and rabbit anti-Eph
(Dearborn et al., 2002
). A
peptide consisting of amino acids 361-612 of Skd was produced in
Escherichia coli with an N-terminal His-tag, purified on Ni-NTA
agarose, and used to raise rabbit polyclonal antibodies (Covance). Antiserum
was used at a final concentration of 1:5000 for tissue staining. A peptide
consisting of amino acids 2040-2229 of Kto was produced in E. coli
with an N-terminal His-tag, purified on Ni-NTA agarose, and used to raise both
rat and guinea pig polyclonal antibodies (Covance). Antiserum was used at a
final concentration of 1:1000 for tissue staining. Fluorescence images were
obtained on a Leica TCS NT confocal microscope. The NIH Image program was used
to measure clone roundness: clones falling entirely within the wing pouch were
outlined on an enlarged Photoshop image and the circularity was calculated as
4
A/L2, where A is the clone area and L is the perimeter
(Lawrence et al., 1999
).
Wild-type measurements were taken from twin spots in the same set of images.
Significance was calculated using a two-tailed t-test. To compare
clone sizes, clones falling entirely within the wing pouch were outlined using
NIH Image; their areas were measured in pixels and normalized to the total
area on the same image of the wing pouch as outlined by Wg staining.
Protein extraction and immunoprecipitation
Proteins were extracted from 0- to 23-hour-old white-
embryos, and from embryos expressing UAS-skd1 and UAS-kto4
under the control of da-GAL4, at 25°C. Embryos were collected
with PBT (0.2% Triton in PBS), dechorionated in 50% bleach, washed with
distilled water and briefly ground in lysis buffer [50 mM Tris-HCl (pH 7.4),
150 mM NaCl, 1 mM EDTA (pH 7.4), 1% Triton X-100, 0.1% SDS] in the presence of
protease inhibitors (1 mM PMSF; 1 µg/ml each of aprotinin, pepstatin and
leupeptin) and phosphatase inhibitors (1 mM NaF, 1 mM
Na2VO4). Homogenates were rocked for 30 minutes and spun
at 13,000 g for 15 minutes at 4°C. Protein amounts in the
supernatant were quantified by the Bradford method to normalize quantities of
extract used for immunoprecipitation between white--, and
skd- and kto-overexpressing embryos. An aliquot of the total
extract was diluted in 5xLaemmli buffer to run on the gel directly.
Extracts were supplemented with lysis buffer to normalize the volume used for
immunoprecipitation (IP). Guinea pig anti-Kto antibody (1:100 final dilution),
or anti-Skd antibody (1:100 final dilution) or no antibody (for control IPs)
was added to the samples, which were then rocked for 2 hours at 4°C.
Protein-A agarose (Roche) was then added and the samples rocked for 2 hours at
4°C. Beads were spun for 30 seconds and washed four times with lysis
buffer, including protease and phosphatase inhibitors, and once with the same
buffer without SDS. Beads were then re-suspended in 2xLaemmli
buffer.
Western blotting
Samples were boiled for 5 minutes at 95°C before running on SDS-PAGE
gels [7% to detect Skd and Kto, or 12% to detect dSOH1 (Trap18
FlyBase)]. Each immunoprecipitation was divided into equal quantities for
detection of Skd, Kto or dSOH1. Wet electrotransfers of gels to nitrocellulose
membranes (Bio-Rad) were performed at 50 mA overnight at 4°C. Blots were
blocked for 2 hours at room temperature in TBT (0.2% Tween 20 in TBS)
containing 10% low-fat milk, and then for 2 hours at room temperature in TBT
containing 10% low-fat milk supplemented with rat anti-Kto (1/5000), anti-Skd
(1/100,000) or anti-dSOH1 (1/10,000) (Park
et al., 2001). Blots were washed four times in TBT and incubated
for 1 hour in TBT containing 10% low-fat milk supplemented with anti-rat,
anti-rabbit or anti-mouse HRP (1/5000 dilution, Jackson Immunoresearch). After
four washes in TBT, blots were developed using the ECL photoluminescence
procedure (Pierce).
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RESULTS |
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|
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Cells lacking skd or kto distort the dorsal-ventral
compartment boundary
The effects of skd and kto on the AP axis appeared to be
limited to alterations of cell affinity. We therefore tested the effects of
loss of these genes on cell affinity at the DV boundary. Because the DV
boundary does not form until the second instar, unlike the AP boundary, which
forms during embryogenesis (Garcia-Bellido
et al., 1973), we could examine the effects of skd and
kto clones generat both before and after boundary formation. When
skd or kto mutant clones that spanned the DV boundary were
generated in first or early second instar larvae before boundary formation,
the entire clone often moved into one of the two compartments, distorting the
compartment boundary (Fig. 2;
Table 1B). For example,
Fig. 2A-D shows a clone located
entirely in the ventral compartment that includes cells expressing the dorsal
selector gene apterous (ap) in its dorsalmost part. These
dorsal cells appear to have moved into the ventral compartment. When the
larger part of the clone was dorsally derived, non-ap-expressing
cells were found in the dorsal compartment
(Fig. 2E-H). The result in each
case was to create a straight boundary between dorsal cells and mutant cells,
or between ventral cells and mutant cells, rather than between dorsal and
ventral cells. Interestingly, wg was still activated at the border
between the ap-expressing and non-expressing cells, so that
wg expression no longer formed a straight line
(Fig. 2B,F). This suggests that
ap is still able to activate fng and Ser expression
in the absence of skd and kto, allowing N activation and
wg expression at the border of the ap expression domain.
|
The skd and kto genes act together
It is striking that the phenotypes of skd and kto
mutations are identical in every respect examined. We wondered whether double
mutants would show a stronger phenotype, revealing redundancy between the two
proteins. In fact, the phenotype of double mutants was indistinguishable from
either single mutant, as judged by the effects of clones on Elav and Ato
expression in the eye disc, and Ci and Wg expression in the wing disc
(Fig. 3)
(Treisman, 2001).
Double-mutant clones failed to respect compartment boundaries, had a rounded
shape and, like single mutant clones, did not affect cell growth or survival
(Fig. 1K;
Fig. 3). This strongly suggests
that the two proteins act as a unit and that neither can function in the
absence of the other.
|
|
|
The Skd and Kto proteins physically interact
The synergy observed when skd and kto are co-expressed,
and the identical phenotypes of single and double mutants, could result from a
physical interaction between the two proteins. To test this possibility, we
generated polyclonal antibodies to Skd and Kto; both were specific, as no
staining was observed within clones mutant for the corresponding gene
(Fig. 5A-F). As expected, both
proteins were present in the nucleus and showed the ubiquitous distribution
previously observed for their transcripts
(Fig. 5 and data not shown).
Skd and Kto appear to associate in vivo, as the two proteins could be
coimmunoprecipitated both from wild-type embryos, and from embryos
overexpressing UAS-skd and UAS-kto from the ubiquitous
driver daughterless (da)-GAL4
(Fig. 5G,H). This may simply
reflect the presence of both proteins in the mediator complex. However, when
Skd and Kto were overexpressed, the amount of each protein
coimmunoprecipitated by the other increased without a corresponding increase
in precipitation of the core mediator component dSOH1
(Fig. 5G,H), which suggests
that Skd and Kto can also associate outside the complex.
|
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DISCUSSION |
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Compartmentalization along the DV axis takes place during the second larval
instar. skd- or kto-mutant clones generated after this time
do not appear to cross the compartment boundary in either direction, which
suggests that loss of skd or kto does not induce a switch
from dorsal to ventral affinity or vice versa. Consistent with this,
skd and kto are not required for the dorsal expression of
Caps, an adhesion molecule that when misexpressed in ventral cells forces them
toward the compartment boundary (Milan et
al., 2001). When skd- or kto-mutant cells are
generated before the compartment boundary has formed, the boundary does not
form a straight line within the clone; instead, an affinity boundary appears
to separate mutant from either dorsal or ventral wild-type tissue. The
bidirectional nature of these distortions again indicates that the mutant
cells have not taken on the affinity of either compartment. skd- or
kto-mutant clones also form straight boundaries with wild-type tissue
in both the dorsal and ventral compartments, and round up to minimize their
contact with wild-type cells. Loss of skd and kto may
prevent the establishment of both dorsal and ventral affinity, or may promote
the acquisition of a novel affinity. N activation is required to establish the
DV boundary; however, alterations in N signaling relocate the Wg stripe to the
border of the mutant clone, rather than to the border of ap
expression (Micchelli and Blair,
1999
; Rauskolb et al.,
1999
). In skd- and kto-mutant clones, Wg remains
at the border of the ap expression domain. If the effects of
skd and kto on affinity are mediated by changes in N
signaling, they must alter transcriptional regulation by N in a way that
leaves wg expression unaffected. This would be consistent with
changes in the expression of other N-regulated genes that we have observed in
skd- and kto-mutant clones (F.J. and J.E.T.,
unpublished).
The adhesion molecules underlying compartment boundary formation have been
notoriously elusive, even in screens specifically designed to identify them
(Vegh and Basler, 2003),
perhaps because multiple molecules each make a small contribution to cell
affinity. Caps and Trn appear to confer some aspects of dorsal affinity on
ventral cells, but loss of both molecules does not cause dorsal cells to cross
into the ventral compartment (Milan et
al., 2001
); thus, if these proteins are involved in
compartmentalization they must be redundant with other signals. We considered
the possibility that the Drosophila Ephrin and Eph receptor might act
downstream of skd and kto to control cell affinity
differences. However, neither Dephrin (Ephrin FlyBase) nor Eph
(Bossing and Brand, 2002
;
Dearborn et al., 2002
;
Scully et al., 1999
) showed
compartment-specific expression in the wing disc, and their expression levels
were unaltered in skd- and kto-mutant clones (data not
shown). In addition, overexpression of wild-type Eph failed to rescue the
boundary crossing behavior of skd-mutant clones (data not shown).
Although we have not been able to identify the crucial adhesion molecules for
boundary formation, our demonstration that they are likely to be among the
target genes of Skd and Kto may aid in their discovery.
Skd and Kto regulate a subset of Ci target genes
Our results imply that Skd and Kto assist Ci to regulate the genes that
confer anterior cell affinity, yet skd and kto are clearly
not required for Ci to activate dpp or ptc. This represents
the first situation in which the effects of Hh on cell affinity are
specifically disrupted without a global effect on Hh signaling. Hh regulates
Ci both by blocking its cleavage to a repressor form, and by converting the
full-length protein to a transcriptional activator and transporting it to the
nucleus (Aza-Blanc et al.,
1997; Chen et al.,
1999a
; Methot and Basler,
1999
; Ohlmeyer and Kalderon,
1998
; Wang and Holmgren,
2000
). Some Ci target genes are thought to be controlled primarily
by the repressor or the activator form, whereas others respond to both
(Methot and Basler, 1999
).
However, both the repressor and activator forms have been shown to act through
common DNA binding sites in a minimal dpp enhancer
(Muller and Basler, 2000
). The
effects of Skd and Kto cannot be specific for the repressor form of Ci, as
hh, which is a target of the repressor form
(Methot and Basler, 1999
), is
not de-repressed in skd- or kto-mutant cells in the anterior
wing disc (data not shown).
skd and kto likewise affect only a subset of Hh target
genes in the eye disc. In skd- or kto-mutant clones in the
eye disc, Hh is still able to activate ato and dpp
expression, although at slightly reduced levels
(Treisman, 2001). However,
expression of another Hh target gene, rough
(Dominguez, 1999
), is lost in
these clones (J.E.T., unpublished). The enhancer sequences mediating Hh
regulation of these genes have not yet been analyzed in detail; it will be
interesting to determine what features of an enhancer make it dependent on Skd
and Kto to recruit the mediator complex. We also do not know whether Skd and
Kto interact directly with Ci, or with other factors binding to the same
enhancer element. Although we cannot detect a stable interaction between Skd
and Ci by immunoprecipitation, it is still possible that a transient
interaction or an interaction with a small proportion of the total Ci protein
occurs in vivo. It is also possible that Skd and Kto do not affect the
activity of Ci directly, but assist a transcription factor downstream of Ci
that activates a subset of its target genes.
TRAP230 and TRAP240 may constitute a submodule of the mediator
complex
Our current and previously published data
(Treisman, 2001) demonstrate
that loss of either skd, kto or both genes has exactly the same
effect, and that the two genes produce a more severe phenotype when
overexpressed in combination. We have also shown here that the Skd and Kto
proteins interact with each other, and it seems that this interaction can
occur outside the mediator complex. These observations strongly suggest that
Skd and Kto function as a unit. Both proteins might interact simultaneously
with transcription factors such as Ci; alternatively, one of the two proteins
might be required to attach the other to the mediator complex. The SUR2,
TRAP100 and TRAP95 subunits of the mouse complex appear to associate as a
submodule, with both SUR2 and TRAP100 required for its incorporation
(Ito et al., 2002
;
Stevens et al., 2002
). TRAP240
and TRAP230 may form another such submodule. Together with Cdk8 and Cyclin C,
they are present only in larger forms of the mediator complex, such as ARC,
TRAP, DRIP or NAT, but not in smaller forms, such as PC2 and CRSP
(Rachez and Freedman, 2001
).
Interestingly, the larger ARC complex fails to promote transcription from an
Sp1 and SREBP-dependent enhancer that is strongly activated by CRSP
(Taatjes et al., 2002
).
Although the roles of individual subunits in promoting this selectivity have
not been defined, it is possible that TRAP230 and TRAP240 could prevent the
mediator complex from acting on certain enhancers while promoting its activity
on others. However, it is also possible that the repressive effect is caused
by phosphorylation of TFIIH by Cdk8
(Akoulitchev et al., 2000
), or
to the absence of Crsp70. Distant homologs of TRAP240, TRAP230, Cdk8 and
Cyclin C, the yeast Srb8-11 proteins, also form a separable submodule of the
mediator complex that can repress transcription by phosphorylating RNA
polymerase II, but that can also phosphorylate activators
(Ansari et al., 2002
;
Borggrefe et al., 2002
;
Boube et al., 2002
;
Hengartner et al., 1998
;
Lee et al., 2000
;
Nelson et al., 2003
;
Song and Carlson, 1998
).
These observations suggest that the mediator complex consists of a core
complex, perhaps PC2/CRSP or a smaller subset of these subunits, and accessory
subcomplexes that interact with specific sets of transcription factors
(Malik and Roeder, 2000). In
Drosophila, mutations in dTrap80 and dMed6 are both
cell-lethal, suggesting that these subunits are essential for crucial
functions of the mediator complex (Boube
et al., 2000
; Gim et al.,
2001
). Loss of any of several core components of the C.
elegans mediator complex causes embryonic lethality
(Kwon et al., 2001
;
Kwon and Lee, 2001
;
Kwon et al., 1999
), whereas
mutations in sur-2, sop-1, encoding a TRAP230 homolog, and
sop-3, encoding a TRAP220 homolog, cause milder defects
(Singh and Han, 1995
;
Zhang and Emmons, 2000
;
Zhang and Emmons, 2001
). Human
TRAP220 has been shown to interact specifically with nuclear receptors
(Hittelman et al., 1999
;
Yuan et al., 1998
), and
knocking out the mouse gene prevents cells from responding to thyroid hormone
and estrogen (Ito et al.,
2000
; Kang et al.,
2002
). It is not known with which transcription factors TRAP230
and TRAP240 interact. In addition to their effects on cell affinity, we have
evidence that skd and kto are required for the expression of
some Wg and N target genes (F.J. and J.E.T., unpublished). These very large
and highly conserved proteins are likely to present a large number of
interaction surfaces, or perhaps even exhibit enzymatic activities. Their
further study will shed light on the functions of the mediator complex and its
interactions with specific developmental signaling pathways.
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ACKNOWLEDGMENTS |
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