Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA
* Author for correspondence (e-mail: camp{at}pitt.edu)
Accepted 19 October 2004
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SUMMARY |
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Key words: Brinker, Drosophila wing, Repression, Imaginal disc, Dpp
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Introduction |
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Our understanding of how a single transcription factor can activate or
repress a gene at one concentration, but is required at higher levels to
influence another, is still fairly rudimentary. Again, some of the best
insights into how threshold responses to transcription factor gradients are
deciphered at the level of cis-regulatory elements have come from studies of
transcription factors acting during early Drosophila embryogenesis.
The most obvious mechanisms involve modifying the number of binding sites or
the affinities of those sites in enhancers to vary their sensitivity to a
transcription factor, and these mechanisms appear to be used in enhancers
regulated by Dorsal, Hunchback, Kruppel and Knirps
(Clyde et al., 2003;
Jiang et al., 1992
;
Langeland et al., 1994
).
However, sensitivity to activation or repression may be achieved in other
ways; for example, sensitivity to repression by Giant can to be modified by
the positioning of binding sites in relation to a promoter
(Hewitt et al., 1999
).
Gradients of transcription factors are used to generate spatial patterns of
gene expression in many other systems, including in the Drosophila
wing, where the transcriptional repressor Brinker is expressed in a
lateral-to-medial gradient in the anterior and posterior halves
(Campbell and Tomlinson, 1999;
Jazwinska et al., 1999
;
Minami et al., 1999
;
Muller et al., 2003
). This
pattern of expression is established by the morphogen Decapentaplegic (Dpp, a
TGF-ß), which is expressed at the center of the AP axis and, following
secretion, becomes distributed in medial-to-lateral gradients in the anterior
and posterior compartments (Blackman et
al., 1991
; Entchev et al.,
2000
; Masucci et al.,
1990
; Teleman and Cohen,
2000
). Dpp, acting through the intracellular signal transducer Mad
(a Smad) and Schnurri (a nuclear zinc finger protein), represses brk
expression, so that the graded expression of brk mirrors that of the
Dpp protein (Marty et al.,
2000
; Muller et al.,
2003
).
Brk functions to repress the expression of genes in the wing that were
originally classified as Dpp targets. In fact, in the absence of both Dpp and
Brk, these targets are still expressed, indicating that Dpp regulates their
expression largely indirectly, through the repression of Brk. These targets
include spalt (sal), optomotor-blind (omb;
bifid, bi FlyBase) and the vestigial quadrant enhancer
(vg-QE, an enhancer recapitulating a portion of the expression of the
vestigial gene) (Campbell and
Tomlinson, 1999; Jazwinska et
al., 1999
; Kim et al.,
1996
; Marty et al.,
2000
), and have nested expression domains centered on the
dpp stripe, so that the vg-QE domain is wider than that of
omb, which is wider than that of sal
(Kim et al., 1997
;
Lecuit et al., 1996
;
Nellen et al., 1996
). As all
three genes appear to be targets of Brk, this pattern of expression can be
explained by a differential sensitivity to Brk, with sal being
repressed by very low levels of Brk, but vg-QE requiring higher levels.
Here, we investigate how Brk represses gene expression, to further our
understanding of why some targets are more sensitive to Brk than others.
Previous studies have suggested that Brk may repress different targets by
different mechanisms. First, Brk-binding sites in the cis-regulatory regions
of some embryonic targets, including zen and Ubx, overlap
with those of an activator, namely Mad (which can function both to activate
and repress at different loci), and in vitro studies indicate that Brk and Mad
can compete for binding to the same region of DNA
(Kirkpatrick et al., 2001;
Rushlow et al., 2001
;
Saller and Bienz, 2001
).
Second, Brk possesses interaction motifs for the co-repressors Groucho (Gro)
(Chen and Courey, 2000
) and
CtBP (Chinnadurai, 2002
),
indicating that it may use more active mechanisms to repress targets
(Hasson et al., 2001
;
Saller and Bienz, 2001
;
Zhang et al., 2001
). Loss of
Gro or CtBP does result in derepression of some Brk targets, such as the
vg-QE, but not others, such as omb, indicating that Brk may use
different mechanisms to repress different genes
(Hasson et al., 2001
).
We show that Brk requires its DNA-binding domain (DBD) plus a repression domain to act as a repressor, the DBD alone is insufficient to repress targets, even those that have overlapping Brk- and Mad-binding sites. This poses the question of whether competition is a real phenomenon in vivo. Brk possesses at least three independent repression domains, the Gro and CtBP interaction motifs (GiM and CiM, respectively) and one other domain, defined here as 3R. However, these domains are not equivalent; 3R is sufficient for repression of omb but not sal, and this difference may be related to the spacing of Brk-binding sites in relation to activator sites. Thus, although sal and omb show quantitative differences in their response to Brk, this may actually be based more on qualitative differences in the mechanisms that Brk uses to repress them.
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Materials and methods |
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Fly strains and mutational analysis
Flies carrying the following existing alleles or transgenes were used:
brkF124, brkE427,
brkF138, brkM68,
CtBP87De-10, groE48, vg-QE
(P{vg(806)-lacZ}), hs-GFP (Avic\GFPhs.T:Hsap\MYC), hs-flp
(P{hsFLP}22), FRT18A (P{ry[+t7.2]=neoFRT}18A), FRT82B (P{neoFRT}82B), Ubi-GFP
(P{Ubi-GFP(S65T)nls}3R), omb-lacZ (P{lacW}biPol-1), C765
(Scer\GAL4C-765), en-Gal4 (P{en2.4-GAL4}e16E), UbxB (P{Bhz}) and
24B-Gal4 (P{GawB}how24B). Unless indicated otherwise in parentheses, all
genotypes are as denoted in FlyBase
(http://flybase.bio.indiana.edu),
where more information on each can be found. To molecularly characterize
mutants, the brk gene was amplified by PCR from genomic DNA from
hemizygous embryos and was sequenced.
Generation of in vitro mutated/modified UAS-brk transgenes
The UAS-brkA438 line, which produces an unmodified, untagged
wild-type Brk protein, was generated by cloning a brk cDNA into a
modified pUAST vector, which had the white gene removed. A >y+> flp-out
cassette was inserted between the UAS sequences and the cDNA. Transgenic flies
were generated by standard procedure and the flp-out cassette was removed from
transformants using hs-flp, to generate UAS-brkA438. All other
transgenes were cloned into the standard pUAST vector and included a sequence
to introduce two copies of the HA tag to the C terminus, the sequence of this
was (GS/EF/RS)MAGNIYPYDVPDYAGYPYDVPDYAG (the HA sequence
is underlined).
The exact locations of mutations and deletions in different transgenes are
indicated in Fig. 2. C-terminal
deletions were generated by PCR using an external primer, and internal primer
flanked by KpnI and either EcoRI, BglII or
BamHI sites, respectively. Mutations and internal deletions were
generated by inverse PCR followed by religation using two internal primers
flanked by restriction sites generating the following changes to sequence. CM
mutation (CiM), PMDLSLG to AMAAALA (NotI); GM mutation (GiM), FKPY to
FAAA (NotI; similar mutations were shown to result in loss of CtBP
and Gro binding to Brk); NA deletion (3R), residues 148-200 to RS
(BglII); A17 deletion, residues 173-189 to RS
(BglII). The NLS and NLSW proteins were generated by PCR using an
internal primer flanked by sequences encoding the following, PPKKKRKV
(matching the NLS sequence from SV40 T antigen
(Kalderon et al., 1984
) plus
WRPW for NLSW. The EC construct was generated by amplifying residues 151-228
and 383-452, respectively, by PCR and cloning onto NLS. More details on each
construct and methods of construction are available on request.
|
Clones in discs were identified by the loss of GFP.
Ectopic expression of UAS-transgenes was achieved by independently crossing transformant lines to two Gal4-expressing lines: en-Gal4 (expressed in the posterior) and C765 (ubiquitous expression in the wing). For assigning activity level as observed in adult wings, the following criteria were used.
++++, wild-type level (no modified/mutated protein achieved this level). No adults were obtained with en-Gal4 even when reared at 17°C. With C765 at 20°C, there was an almost complete loss of wing blade.
+++, some adults were obtained with en-Gal4 at 20-25°C, but showed substantial loss of posterior wing tissue and veins. With C765 at 25°C, there was an almost complete loss of wing blade.
++, adult flies were obtained with en-Gal4 at 25-30°C. Their wings had a loss of tissue or a fusion of veins IV and V and loss of the posterior crossvein. With C765 at 30°C, the wings were slightly smaller and had vein defects, including extra crossveins.
+, adult flies obtained with en-Gal4 at 25-30°C, with loss of the posterior cross vein. With C765 at 30°C there was little or no effect on the wings.
, no activity. No abnormal phenotype under 25°C; at 30°C there was often some disruption to wing venation, such as extra small veins around the posterior crossvein and vein V. This was distinct from the other phenotypes above and may be caused by weak dominant-negative activity.
At least three lines of each construct were tested apart from F124 (one line). Although there was some variability in the level of activity from line to line, in general most lines from any one construct fell into the same category of activity level. To be assigned to one of the above categories, at least two lines from a construct had to have a similar level of activity; in fact, for most constructs, at least three lines had similar levels of activity.
Generation of mutations in UAS-brkA438
A homozygous strain of UAS-brkA438 was mutated with ethylmethane
sulphonate (EMS) using standard procedure
(Grigliatti, 1986) and crossed
to the ubiquitous Gal4 line C765. The progeny were raised at 25°C, which
normally results in flies with almost no wing blade
(Fig. 3B); flies with larger
wings were selected. The UAS-brk transgene was amplified from potential
mutants by PCR and sequenced.
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Results |
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The activity of these mutated/modified Brk proteins was compared with that
of the wild-type protein by analyzing the expression of known Brk targets:
sal, omb and the vg-QE in wing discs, and a Ubx reporter, UbxB, in
embryos (Kirkpatrick et al.,
2001; Saller and Bienz,
2001
). For endogenous mutants (brkF124,
brkF138 and brkE427), expression of
these targets was examined in marked homozygous mutant clones. For null
mutants, such clones show misexpression of Brk targets in lateral regions
(Campbell and Tomlinson, 1999
;
Jazwinska et al., 1999
;
Minami et al., 1999
).
The UAS-brk transgenes were misexpressed in the posterior of wing discs using en-Gal4, and the expression of Brk targets in the posterior was compared with that in the anterior. Larvae were raised at different temperatures to vary the amount of protein ectopically expressed (Gal4 is cold sensitive). At 20°C, wild-type Brk3PF3 completely repressed sal and omb, and almost completely repressed the vg-QE (Fig. 2D, Fig. 5A,J). At 17°C, sal is still completely repressed, but some omb expression can be detected, indicating that wild-type Brk is more effective at repressing sal than omb (Fig. 5B). In embryos, UAS-brk transgenes were misexpressed in mesoderm using the 24B driver; at 25°C, Brk3PF3 completely repressed UbxB (Fig. 6B).
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Mutations in the DBD reduce or completely eliminate Brk activity
Mutations in the DBD were identified in one of the endogenous mutants,
brkF124, and four of the UAS-brkA438 mutants,
D44, S4, C and F2 (Fig. 2).
brkF124 has an amino acid substitution in the recognition
helix R82W (Fig. 2B), and
resulted in a protein with little or no activity: brkF124
mutant clones in wing discs were indistinguishable from those of null alleles,
showing autonomous misexpression of sal, omb and the vg-QE
(Fig. 4A,B). The mutations in
UAS-brkA438 were located in different regions of the DBD
(Fig. 2C) and had varying
effects on the activity of the resulting Brk protein, as assessed by the size
of the wing produced with C765 (Fig.
3), although none appeared to completely eliminate activity. We
also generated UAS-brkF124, which generated a full-length,
tagged protein with the same mutation that is found in
brkF124. This also had no repressor activity, as assessed
by sal and omb expression, although when misexpressed at
high levels it did result in the formation of ectopic veins in adult wings,
which may be due to an abnormal activity of this protein; for example, it may
have a modified DNA-binding specificity
(Fig. 2D,
Fig. 7F).
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The in vitro studies suggesting that Brk may use binding site competition
as a mechanism for repression were carried out with genes expressed in the
embryo (Kirkpatrick et al.,
2001; Rushlow et al.,
2001
; Saller and Bienz,
2001
), and it is possible that Brk does not use this mechanism in
the wing. Consequently, we tested one of these embryonic targets, UbxB
(Thuringer et al., 1993
),
which is expressed in the midgut mesoderm, but found that it too was not
repressed by BrkNLS (Fig.
6C).
Addition of a minimal repressor motif to the DBD restores activity
One possible reason for the inactivity of the BrkNLS protein is
that it cannot actually bind DNA in vivo. To test this, we designed an
identical construct, UAS-brkNLSW, with the addition of a minimal
repressor motif consisting of just four amino acids, WRPW
(Fig. 2). WRPW functions as a
repression motif by recruiting Gro (Aronson
et al., 1997; Fisher et al.,
1996
). BrkNLSW protein has considerable activity; it
significantly reduced the size of adult wings (but is not as effective as
wild-type protein), and it could repress sal completely and
omb almost completely (Fig.
5F). In this regard it behaves similarly to wild-type protein,
i.e. it represses sal more effectively than omb. It had no
effect, however, on vg-QE expression (Fig.
5K), but could substantially repress UbxB in the embryo
(Fig. 6D).
The CiM and GiM are required for Brk to repress some, but not all targets
If the DBD alone is unable to repress gene expression, other regions of the
protein must be required, the obvious candidates being the CiM and GiM.
However, analysis of the brkF138 mutant, which lacks both
motifs, revealed that Brk could repress at least one target, omb, in
their absence. This mutant is predicted to produce a protein truncated at
residue 332, i.e. before these two motifs
(Fig. 2B). In contrast to null
clones, omb was not derepressed in brkF138 clones
(Fig. 4C,D), although we cannot
rule out the possibility that there may be a very minor expansion of
expression of one or two cell diameters (the edge of the endogenous domain is
difficult to resolve precisely, because it is not completely straight).
However, sal and the vg-QE were ectopically expressed in some
brkF138 clones (Fig.
4C,D,E). For sal, this ectopic expression was restricted
to the omb domain, i.e. expansion of the sal domain did not
extend to the edge of the wing pouch, as it would in brk null mutant
clones.
A recent study showed that sal expression is dependent upon Omb
(del Alamo Rodriguez et al.,
2004), suggesting that the lack of ectopic sal expression
in lateral brkF138 clones (i.e. outside of the
omb domain) could be explained by the absence of ectopic omb
in these clones, rather than by BrkF138 directly repressing
sal. In support of this, sal is not misexpressed in omb
brk null double-mutant clones (data not shown). del Alamo Rodriguez et
al. also suggested that vg-QE expression is dependent upon Omb
(del Alamo Rodriguez et al.,
2004
). However, vg-QE expression actually shows expansion into
lateral brkF138 clones
(Fig. 4E), i.e. in the absence
of any ectopic omb expression. Possible explanations for these
conflicting results are that vg-QE expression may not be dependent upon Omb in
all situations, or that there is some ectopic omb expression in these
clones but it cannot be detected with the omb enhancer trap used.
CtBP or Gro is required for repression of sal
BrkF138 protein appears to be able to repress omb
without recruiting either CtBP or Gro, but either one or both may be required
for repression of sal and the vg-QE. This is consistent with previous
studies showing that neither CtBP nor Gro is required for the repression of
omb, and that Gro is required for repression of the vg-QE
(Hasson et al., 2001). We have
extended these studies, and have found that, in contrast to omb, Gro
is required for the full repression of sal, whereas CtBP can
partially substitute for the loss of Gro [this is in contrast to previous
claims that neither Gro nor CtBP is required for repression of sal
(Hasson et al., 2001
)].
CtBP/gro double mutant clones in the wing had the same
phenotype with respect to sal expression as
brkF138 clones, i.e. ectopic expression, but only within
the omb domain (Fig.
4F). By contrast, sal expression is normal in
CtBP single mutant clones (Fig.
4G), demonstrating that the ectopic expression of sal
found in CtBP/gro double mutant clones can be rescued
completely by Gro alone. sal expression in gro single mutant
clones, however, is occasionally expanded
(Fig. 4H), but not as
extensively as in the CtBP/gro double mutant clones,
indicating that CtBP can provide some, but not full, repressor activity to
narrow the lateral limit of sal expression.
CtBP/Gro-independent repression by Brk is dependent upon a domain, 3R, located just C-terminal to the DBD
The inactivity of BrkNLS in comparison to BrkF138
suggested that the region between the DBD and the CiM contains an additional
repression domain. This was tested by misexpression of additional modified Brk
proteins. BrkStop1 is similar to BrkF138, being
truncated immediately before the CiM (Fig.
2), and was tested initially to rule out the possibility that any
Brk activity in the brkF138 mutant was due to read through
of the stop codon. Misexpression of BrkStop1 in the posterior
compartment at 25°C resulted in complete repression of omb and
sal, and of the vg-QE (Fig.
5H,L). At 20°C (with concomitant lower expression of the
transgene), omb was still almost completely repressed, although there
was still weak residual expression remaining in the posterior compartment
(Fig. 5I). By contrast, at this
temperature sal was still strongly expressed in the posterior
(although the size of its domain was significantly reduced), demonstrating
that BrkStop1 is much more effective at repressing omb
than sal, and, thus, that it differs from the wild-type protein.
Although we cannot rule out the possibility that BrkStop1
represses sal directly 25°C, it is likely that it does this
indirectly: sal requires Omb for expression
(del Alamo Rodriguez et al.,
2004) and omb is completely repressed at this temperature
(Fig. 5H). It should be pointed
out, however, that at 20°C, omb expression cannot be detected in
some cells that are expressing sal
(Fig. 5I). Because previous
studies (del Alamo Rodriguez et al.,
2004
), and our own (not shown), indicate that sal
expression is absolutely dependent upon Omb, the likeliest explanation for
this observation is that omb is expressed in these cells at levels
sufficient for expression of sal, but that is just too low to be
detected with the omb-lacZ line.
BrkStop1 can also repress the reporter UbxB and the vg-QE. It
can, in fact, repress UbxB even more effectively than BrkNLSW does
(Fig. 6E). It is unclear why
BrkStop1 can repress the vg-QE when BrkF138 appears to
be compromised in this respect (Fig.
4E), and when vg-QE expression is upregulated in gro
mutant clones (Hasson et al.,
2001). It is possible that Gro is required for repression of the
vg-QE when Brk is present at physiological levels, but not at the higher
levels that can be achieved with the UAS/Gal4 system.
Additional truncations identified a minimal protein, BrkA,
truncated at residue 206 (Fig.
2D), with similar activity to BrkStop1. A series of
additional constructs, which either had repressor activity, BrkEC
and BrkStop117, or did not, BrkStop1NA and
BrkA2, identified the region 151-206 as being the minimum region
sufficient to confer repressive activity. This region has been termed 3R, for
the third repression domain, in addition to the CiM and GiM
(Fig. 2A). 3R consists of a
histidine-rich region, a stretch of poly-alanine and some unique sequence at
the C terminus. Further studies are required to narrow down essential
sequences in this region; for example, the fact that
BrkStop1
A17 still has considerable activity suggests that
the stretch of poly-alanine is probably not essential.
Brk protein possessing only the CiM has significant activity
Experiments described above show that a Brk protein possessing only a GiM,
or 3R, has significant activity (Fig.
2). We further demonstrated that a protein,
BrkStop1NAC, possessing only a CiM (deleting the 3R domain and
terminating immediately after the CiM; Fig.
2D) repressed omb and sal, and significantly
reduced the size of adult wings (Fig.
2D, Fig. 5G). It
behaved similarly to wild-type Brk, and was more effective at repressing
sal than omb.
Inactivation of a single repression domain/motif reduces the activity of Brk
To test whether the loss of an individual repression domain/motif resulted
in reduced Brk activity, we tested the activity of Brk proteins in which
either the CiM (BrkCM), GiM (BrkGM) or 3R
(BrkNA) was mutated or deleted. In contrast to wild-type
Brk3PF3 (Fig. 7B),
adult flies did survive following the misexpression of each of these single
mutant transgenes with en-Gal4 at 20°C
(Fig. 7C) indicating that they
were less active than the wild-type protein. In terms of gene expression, each
could repress sal, omb and the vg-QE, but were essentially too
similar in their activity level to each other, and to the wild-type
Brk3PF3 protein, to make any clear conclusions.
Brk must possess an additional repression domain(s)
If Brk requires a DBD plus a repression domain to function, and 3R, CiM and
GiM are the only repression domains/motifs, then mutation or deletion of all
three should render the Brk protein inactive. However, in Brk3M, 3R
is deleted, and the CiM and GiM are mutated, but this protein still had
significant activity (Fig. 2D,
Fig. 7D), indicating that there
must be at least one more repression domain/motif, which is probably located
between the CiM and the C terminus. Preliminary studies indicate a fourth
repression domain may lie between the CiM and GiM, but further analysis is
required to confirm this finding (not shown).
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Discussion |
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Brk does not appear to repress by simple competition
The simplest method of transcriptional repression involves competition with
an activator, and can operate at the level of DNA if the activator and the
repressor have the same, or overlapping, binding sites in an enhancer. In
theory, assuming a transcription factor is nuclear, it should only require a
DNA-binding domain to act in this fashion. Brk has been shown to possess an
N-terminal sequence-specific DNA-binding domain (DBD;
Fig. 1)
(Kirkpatrick et al., 2001;
Rushlow et al., 2001
;
Saller et al., 2002
;
Sivasankaran et al., 2000
;
Zhang et al., 2001
), and here
we have identified several mutations in this domain that either completely
inactivate or reduce the activity of the protein (Figs
2,
3,
Fig. 4A), indicating that this
region is essential for Brk activity.
Previous studies suggested that Brk could function by competition, more
specifically, by competing with Mad for overlapping binding sites in vitro
(Kirkpatrick et al., 2001;
Rushlow et al., 2001
;
Saller and Bienz, 2001
).
However, a nuclear localized Brk protein consisting primarily of the DBD,
BrkNLS, cannot repress any Brk target in vivo
(Fig. 2D,
Fig. 5C,
Fig. 6C), including the
embryonic UbxB reporter, which has been shown to possess overlapping Brk and
Mad binding sites that Brk and Mad can compete for in vitro
(Kirkpatrick et al., 2001
;
Saller and Bienz, 2001
). It is
possible that BrkNLS cannot bind to DNA in vivo. However, a
modified protein, BrkNLSW, which is identical to BrkNLS
apart from the addition of the four amino acids WRPW
(Fig. 2D) that recruit the
co-repressor Gro (Aronson et al.,
1997
; Fisher et al.,
1996
), can repress targets
(Fig. 5F,
Fig. 6D), indicating that
BrkNLS should also be capable of binding to these targets in
vivo.
Competition has been proposed as a mechanism for many transcriptional
repressors. However, direct in vivo support for or against such proposals is
rare, at least of the sort presented here, i.e. testing, in vivo, the ability
of a protein consisting largely of a functional DBD, which has access to the
nucleus, to repress a target for which there is in vitro evidence for
overlapping binding sites with an activator. There is some in vivo evidence
that the Drosophila embryonic repressor Kruppel can repress a
synthetic enhancer containing overlapping binding sites with the activators
Dorsal and Bicoid (Nibu et al.,
2003). However, although this repression was CtBP-independent, and
further studies are required to rule out additional domains outside of the DBD
being required (Licht et al.,
1990
) in a similar fashion to the 3R domain in Brk. The paucity of
good examples of binding-site competition in vivo in eukaryotes is in stark
contrast to in prokaryotes (Ptashne and
Gann, 2001
), and raises the question of how common this phenomenon
really is in eukaryotes.
Brk possesses at least three independent repression domains/motifs
If Brk cannot repress by competition it must possess repression
domains/motifs, and previous studies identified interaction motifs for the
co-repressors CtBP and Gro (CiM and GiM)
(Hasson et al., 2001;
Saller and Bienz, 2001
;
Zhang et al., 2001
). However,
repression of at least one Brk target, omb, was previously shown not
to require CtBP or Gro (Hasson et al.,
2001
). This is consistent with our demonstration that the protein
produced by the endogenous mutant brkF138, which is
truncated before the CiM and GiM, can still repress omb
(Fig. 4D). Truncated proteins
that lack the CiM and GiM, BrkStop1, BrkEC and
BrkA (Fig. 2D), can
also repress omb (Fig.
5H), but only if they contain a specific region between the DBD
and CiM that has been classified as a third repression domain, 3R
(Fig. 2A). Further studies are
required to determine if 3R is a true autonomous repression domain, i.e. if it
can function outside of Brk, or if it is more specific (for example,
antagonizing activators such as Mad), and to determine what its specific
properties are (for example, how close do Brk sites have to be to activator
sites for 3R to be effective?).
Differential activity of the repression domains
The three repression domains/motifs of Brk are not equivalent
(Fig. 2D). Wild-type Brk and
proteins containing only a GiM, BrkNLSW, or only a CiM,
BrkStop1NAC, can repress both sal and omb, and
they are more effective at repressing sal than omb
(Fig. 5A,B,F,G). Analysis of
gro and CtBP single and double mutant clones revealed that
Gro is required for normal repression of sal in wing discs, and that
CtBP can provide some, but not always complete, activity for the repression of
sal in the absence of Gro (Fig.
4F-H). By contrast, Gro and CtBP are not required for repression
of omb (Hasson et al.,
2001).
The 3R domain is sufficient for Brk to repress omb
(Fig. 4D,
Fig. 5H) and the UbxB enhancer
in embryos (Fig. 6E), but is
deficient for the repression of sal
(Fig. 4D). Furthermore,
misexpression of proteins possessing only the 3R domain (plus the DBD) are
much more effective at repressing omb than sal, i.e. the
converse of wild-type Brk or Brk possessing only a GiM or a CiM
(Fig. 5I). Although some
results suggested that 3R may confer a limited ability to repress sal
(Fig. 5H), this is probably
indirect, because a previous study (del
Alamo Rodriguez et al., 2004), and our own (not shown),
demonstrated that sal requires Omb to be expressed, and if
omb is repressed directly, sal will be lost also. However,
we cannot completely rule out the possibility that high levels of proteins
possessing only the 3R domain can repress sal directly.
Contradictory results were obtained regarding the ability of 3R to repress the vg-QE. Expression of the vg-QE did show expansion in some brkF138 clones (Fig. 4E), indicating that the truncated protein produced in this mutant (which only has the 3R repression domain) cannot efficiently repress this enhancer. However, similar in vitro truncated proteins, such as BrkStop1, could efficiently repress vg-QE expression when misexpressed using the UAS/Gal4 system (Fig. 5L). Such a difference could simply be a reflection of the high levels of expression achieved with the Gal4/UAS system, and that, at physiological levels, the 3R domain is not sufficient for complete repression of the vg-QE.
Whether a single repression domain is sufficient for Brk to repress a
particular target may depend upon the positioning of Brk sites in relation to
activator sites (or possibly the promoter) at that target. The UbxB reporter
has overlapping Brk and activator (Mad) sites
(Kirkpatrick et al., 2001;
Saller and Bienz, 2001
).
Analysis of an omb enhancer revealed that an important Brk site may
also overlap with an activator
(Sivasankaran et al., 2000
).
Conversely, analysis of the cis-regulatory elements of the sal gene
indicate that activator and Brk sites are separated
(Barrio and de Celis, 2004
).
Proteins possessing only 3R can repress UbxB and omb, but not
sal, suggesting that 3R may only be sufficient for the repression of
genes in which the Brk sites are situated very close to activator sites.
Multiple repression domains
Why does Brk possess at least three, probably four, independent repression
domains/motifs? There are two obvious answers: qualitative, different
repression domains/motifs are required for the repression of different
targets; and quantitative, more domains/motifs provide greater repressor
activity. Other transcription factors have multiple repression domains and
there is evidence that they have these for either qualitative or quantitative
reasons, and, in some cases, both. For example, in the Drosophila
embryo, the pair-rule protein Runt requires Gro for the repression of one
stripe of the pair-rule genes, even skipped (eve) and
hairy, but not for the repression of engrailed
(Aronson et al., 1997). The gap
protein Knirps represses different stripes of eve; for stripes 4 and
6 it requires CtBP, but for stripes 3 and 7, it does not. However, this
appears to be a quantitative difference, because increasing the levels of
Knirps allows it to repress stripes 4 and 6 even in the absence of CtBP
(Struffi et al., 2004
).
Similarly, Gro appears to increase the repressor activity of the Eve protein
(Kobayashi et al., 2001
).
As discussed above, there is some difference in the ability of the three
domains/motifs in Brk to repress different targets. For example, the 3R domain
is sufficient for the normal repression of omb but not sal.
However, either the CiM or GiM appear to be sufficient for the repression of
both sal and omb (Fig.
2D, Fig. 5F,G), so
why does Brk need the 3R domain? In the absence of Gro and CtBP, the Brk
protein appears fully active in its ability to repress omb, and
recruiting Gro and CtBP does not seem to increase its activity towards
omb; otherwise, the width of the omb domain would be
expected to shift in brkF138 mutant cells, which have no
CiM or GiM, or in CtBP gro double mutant cells, but it does not
(Hasson et al., 2001). It is
possible that, in regard to omb, the 3R domain is more efficient than
either of the other two and provides Brk with sufficient activity to establish
the omb domain in the correct position.
Brk needs to recruit either CtBP or Gro for the repression of some targets,
including sal (Fig.
3E-G) and brk itself
(Hasson et al., 2001), or just
Gro for some others, including the vg-QE
(Hasson et al., 2001
).
Consequently, why does Brk need to recruit CtBP? Mutation of the CiM alone, in
common with mutation or deletion of just the GiM and 3R, does reduce activity
of Brk, as judged by its effect when misexpressed
(Fig. 2D). However, there is no
evidence that CtBP is required specifically for the repression of any Brk
target in the wing, because CtBP mutant clones have no effect on the
expression of any known Brk target in the wing
(Fig. 4G) (Hasson et al., 2001
). The
CtBP and Gro motifs in Brk have been conserved over millions of years
(Fig. 1), and thus, recruiting
CtBP is presumably important for Brk activity. It is possible that CtBP is
required outside of the wing for example in the embryo
(Hasson et al., 2001
)
or for some other, as yet, uncharacterized targets in the wing.
Recruiting both CtBP and Gro does appear to be a little illogical from what
is known about their basic properties, CtBP acting only over a short range,
while Gro acts over much longer ranges. It might be assumed that different
transcription factors would use either Gro or CtBP
(Zhang and Levine, 1999),
because the primary advantage of recruiting CtBP is that it would allow a
transcription factor to repress one enhancer without disrupting the activity
of one nearby, which would be repressed if Gro was recruited, although this
simple model does not always hold (Nibu et
al., 2001
). Consequently, most transcription factors do recruit
only one of these co-repressors. However, there are two other exceptions,
Hairy and Hairless. In Hairy it appears that CtBP may actually be functioning
to antagonize Gro activity and not in its standard role as a co-repressor
(Phippen et al., 2000
;
Zhang and Levine, 1999
). There
is no evidence that it does this in Brk, where it can provide repressor
activity. For Hairless, there is genetic evidence that both CtBP and Gro
provide repressor activity to the protein
(Barolo and Posakony, 2002
),
although it is not clear if CtBP is required to increase the general activity
of Hairless, or for repression of specific targets that cannot be repressed
adequately by Gro.
With the exception of the brkF138 mutant, our analysis has been limited to analyzing the effects of misexpressing modified Brk proteins in positions where the endogenous protein is not found. Consequently, further insights into the precise roles of individual repression domains will require replacing the endogenous gene with one in which only one or two repressions domain have been mutated or deleted.
Thresholds
To conclude, it is often assumed that the sensitivity of one enhancer to a
transcription factor compared with that of another enhancer is based largely
upon the number or the affinity of the binding sites for that transcription
factor in each enhancer. However, other factors are also important; for
example, the ability of the Giant transcription factor to repress a promoter
is related to how closely it binds (Hewitt
et al., 1999). Here, we have shown that the two best characterized
outputs of the Dpp morphogen gradient, sal and omb, appear
to be regulated differently by Brk. Consequently, simply counting binding
sites and measuring their affinity will not reveal why one is more sensitive
to Brk than the other, and we need to factor in what specific repressive
mechanisms are being used, and the relative efficiencies of each.
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ACKNOWLEDGMENTS |
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