1 Department of Microbiology and Immunology, Kimmel Cancer Institute, Thomas
Jefferson University, Philadelphia, PA 19107, USA
2 Department of Biochemistry and Molecular Biophysics, Columbia University, New
York, NY 10032, USA
* Author for correspondence (e-mail: Jim.Jaynes{at}mail.tju.edu)
Accepted 7 November 2002
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SUMMARY |
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Key words: Drosophila, Embryogenesis, Homeodomain, Transcription, Repressor, Cofactor, Pbx, Meis
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INTRODUCTION |
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En carries out these functions, at least in part, by repressing downstream
target genes (Smith and Jaynes,
1996; Tolkunova et al.,
1998
). It has two well-characterized repression domains, one of
which binds the Groucho co-repressor complex
(Han and Manley, 1993
;
Jaynes and O'Farrell, 1988
;
Jaynes and O'Farrell, 1991
;
Jimenez et al., 1997
;
Tolkunova et al., 1998
). Like
many eukaryotic DNA-binding proteins, the specificity of DNA binding by En
alone appears to be lower than that required for highly selective interaction
with specific target genes. This suggests that, like many other DNA binding
proteins, En interacts with cofactors that increase its binding specificity.
En has been shown to be capable in vitro of binding cooperatively to DNA with
the Extradenticle protein (Peltenburg and
Murre, 1996
), although a role for this interaction in vivo has not
been demonstrated.
The extradenticle (exd) gene is also a
homeobox-containing gene that was initially characterized as a mutation
causing multiple homeotic transformations in Drosophila, without
affecting the expression patterns of the homeotic genes. These observations
suggested that it might serve as a cofactor for Hox gene products
(Peifer and Wieschaus, 1990).
Homeotic transformations are seen in zygotic mutants but, when the maternal
contribution to exd function is also removed, embryos show
alterations that suggest a loss of en function, including a loss of
en gene expression, at later embryonic stages
(Peifer and Wieschaus, 1990
;
Rieckhof et al., 1997
).
Molecular studies have confirmed the role of Exd as a cofactor for Hox
proteins, including a function in altering their specificity of binding to DNA
(reviewed in Mann and Chan,
1996
). This function appears to be conserved in the mammalian
homologs of Exd, the Pbx proteins
(Peltenburg and Murre, 1997
;
Phelan et al., 1995
).
A third homeobox gene, Meis1, was discovered as an ecotropic
retroviral insertion site in mice (Moskow
et al., 1995), where its overexpression in conjunction with a
subset of Hox proteins leads to leukemic transformation of hematopoietic stem
cells (Nakamura et al., 1996
).
Meis1 is a mammalian homolog of the Drosophila homothorax
gene (hth), which has been shown to regulate the nuclear localization
of Exd (Rieckhof et al., 1997
)
and to form complexes with Hox proteins, including co-complexes with Exd
(Ryoo et al., 1999
). As with
the cofactor functions of Exd, the interactions and functions of the Meis1
family appear to be largely conserved between Drosophila and mammals
(Jacobs et al., 1999
;
Liu et al., 2001
;
Saleh et al., 2000
;
Shanmugam et al., 1999
;
Shen et al., 1999
). In
mammals, a Hox-class (Antp-class) protein that is part of an endodermally
expressed ParaHox cluster (Brooke et al.,
1998
; Leonard et al.,
1993
) has also been shown to interact functionally with mammalian
homologs of Exd and Hth (Swift et al.,
1998
).
In this work, we show that En interacts specifically with Exd and Hth in vitro and in cultured cells, that Exd and En mediate co-operative repression in cultured cells, and that the genetic functions of both exd and hth are crucial to the repression activity of En in embryos. We identify sloppy paired (slp) as a direct target gene of En, and show that exd and hth are required for En effectively to repress both slp and a second key target gene, wg, in developing embryos. The involvement of exd and hth function in direct repression by En, in conjunction with the observed molecular interactions, suggest that they are likely to participate in En repression complexes in vivo.
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MATERIALS AND METHODS |
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Glutathione-S-transferase (GST) pull-down assays were performed as
described previously (Tolkunova et al.,
1998). GST-En contains En amino acids 1-544 in pGEX-5X-1;
full-length Exd was transcribed from pET15b-Exd, which contains the
complete Exd ORF. Full-length Meis1b was expressed from
pET28b-Meis1b (Steelman et al.,
1997
).
Cell culture assays
Transfections of Schneider line 2 (S2) cells were performed as described
previously (Jaynes and O'Farrell,
1991). Plasmids used for Fig.
5C were pRM-HA3-hth (encoding full-length Hth, 3 µg per 60 mm
plate), pAc-en (Jaynes and O'Farrell,
1988
) (8 µg per plate), pPAc-His6Exd
(encoding full-length Exd tagged at its N-terminus, driven by the
actin5C promoter, 8 µg per plate) and pCaSpeR-hs as filler (to 19
µg total DNA per plate). Nuclear extracts for co-immunoprecipitation (coIP)
experiments were performed as described by Han and Manley
(1993
). For coIPs, antibodies
were immobilized on either Protein-A/agarose or Protein-G/sepharose in Buffer
A (20 mM HEPES pH 7.9, 150 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM
DTT, 0.5 mM PMSF, 10% glycerol), blocked with non-transfected nuclear extract
for 4 hours, incubated with transfected nuclear extract overnight, washed
extensively at 23°C with Buffer A plus 0.2% Triton X-100, eluted in
Laemmli buffer, and analyzed by western blotting essentially as described by
Han and Manley (Han and Manley,
1993
), except that specific proteins were visualized using the ECL
detection system (Amersham). All incubations were at 4°C unless otherwise
indicated.
Plasmids used for Fig. 6
were: (1) pT3CM6-CAT, a chloramphenicol
acetyltransferase (CAT)-expressing plasmid that can be activated by
glucocorticoid receptor (GR) and is derived from pT3N6D-33CAT
(Jaynes and O'Farrell, 1991)
by replacing the N6 homeodomain binding sites with a tandem array of six
composite En-Exd binding sites (Peltenburg
and Murre, 1996
) or by mutated versions of this site (as indicated
in the text and in the legend for Fig.
6); (2) pTAT3-CAT
(Jaynes and O'Farrell, 1991
),
the equivalent plasmid without En-Exd sites; (3) pRK232
(Heemskerk et al., 1991
), an
En expression construct; (4) pRM-HA3-hth, a metallothionein-promoter-driven
Hth expression construct; and (5) pLac82SU
(Dorsett et al., 1989
) as a
reference. The metallothionein promoter was induced by adding CuCl2
to 0.7 mM 24 h after transfection; the GR was activated by adding
triamcinolone acetonide to 10-7 M 48 hours after transfection.
Fig. 6A used 1 µg each of
pRK232 and either pTAT3-CAT or pT3CM6-CAT, as
indicated (without or with En-Exd sites, or with mutated sites), 0.5 µg
pRM-HA3-hth, and 0.5 ng pLac82SU per 60 mm culture dish.
Fig. 6B used 1 µg each of
pT3CM6-CAT and pRK232, 0.04 µg pPAc-GR
(Jaynes and O'Farrell, 1991
),
and 0.5 ng pLac82SU per 60mm culture dish, and either 0.5 (`low') or 1 µg
(`hi') of pRM-HA3-hth where indicated in the figure. Total DNA was normalized
to 6 µg per dish in both Fig.
6A and Fig. 6B.
Fig. 6C used 0.36 µg
pT3CM6-CAT, 0.5 µg pRM-HA3-hth, 0.014 µg
pPAc-GR and 0.18 ng pLac82SU per 35 mm culture dish, and either
0.108 (`low') or 0.36 µg (`high') pRK232, as indicated in the figure. Total
DNA was normalized to 2.16 µg per dish. Three hours prior to transfection,
each dish was treated as described by Clemens et al.
(Clemens et al., 2000
) with 24
µg of double-stranded RNA (dsRNA) encoding either Rad9 or Exd.
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Drosophila stocks and assays
In situ hybridization was performed as described
(Tautz and Pfeifle, 1989).
Antibody staining with anti-lacZ, anti-En, anti-Exd
(Rieckhof et al., 1997
) and
female-specific anti-Sex-lethal antibodies
(Bopp et al., 1991
) was
performed as described previously
(Kobayashi et al., 2001
).
To generate hth mutants with hs-En
(Heemskerk et al., 1991),
P[hs-En E3D]; hthP2/TM3 Sb ftz-lacZ strains were
self-crossed; as controls, P[hs-En E3D]; TM3 Sb ftz-lacZ/+
strains were self-crossed.
To generate exd maternal mutant embryos carrying hs-En,
exd1 FRT 18D/FM7-B1 females were crossed with
OVOD2 FRT 18D; hs-Flp1 males to generate
exd1 FRT 18D/OVOD2 FRT 18D; hs-Flp1/+
females (non-B), which were heat shocked during the wandering larval
stage (1 hour at 37°C every 12 hours) to induce mitotic recombination.
These females with homozygous mutant germ-line clones were then crossed with
either P[hs-En E5] (on the third chromosome) or wild-type (control)
males. The exd1 allele (a.k.a.
exdXP11) is amorphic
(Rauskolb et al., 1995).
To generate exd maternal mutant embryos carrying UAS-en
(Tabata et al., 1995),
exd1 FRT 18D/FM7-B1;; UAS-en females
were crossed with OVOD2 FRT 18D; hs-Flp1;
UAS-en males to generate exd1 FRT 18D/OVOD2
FRT 18D; hs-Flp1/+; UAS-en females (non-B),
which were heat shocked as above to induce mitotic recombination. These
females were then crossed with prd-Gal4/TM3 Sb hb-lacZ males.
Cuticle preparations were performed as described previously
(Fujioka et al., 1999). For
Fig. 4, embryos were collected
and aged for 22 hours at 25°C. Cuticles were prepared and categorized by
severity of defects as follows. The number of abdominal denticle bands fused
was determined in at least 120 embryos (for each data point) of the genotypes
indicated in Fig. 4 and its
legend. The categories, from least to most severe, had the following number of
denticle bands fused (two bands completely fused together were counted as two
bands fused, partial fusions were counted as one band fused): 1-3, 4, 5, 6,
7-8.
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RESULTS |
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First, we expressed En ubiquitously using a heat shock promoter-driven
transgene. This allowed us to focus on a direct target gene of En, based on
the immediate response to ectopic En expression. We examined the responses of
slp and wg, which are repressed by en, and of the
en gene, which is ectopically activated by ubiquitously expressed En
(Heemskerk et al., 1991). The
gene that responded most rapidly to En was slp, which was noticeably
repressed relative to control heat-shocked embryos within 7 minutes of a 5
minute heat pulse (data not shown). Such a rapid response makes it very likely
that the response is direct (Manoukian and
Krause, 1992
; Saulier-Le Drean
et al., 1998
), and strongly suggests that slp is a direct
target of repression by En.
To determine whether repression of slp by En was affected by a
loss of hth or exd function, we examined the effect of the
strong hth allele hthP2
(Kurant et al., 1998) in
combination with a hs-En transgene. As shown in
Fig. 1A-E, the loss of
hth function clearly reduced the repression of slp by En.
This repression of slp expression persisted to later stages of
embryogenesis (data not shown), although the degree of persistence varied from
embryo to embryo because of the relatively mild induction of ectopic En used
here. This mild induction resulted in a level of expression comparable to or
less than that in the endogenous En stripes. We also tested whether loss of
hth function affected the expression of En from the hs-En
transgene by staining embryos with En-specific antiserum. We detected no
difference in the strength or persistence of the ectopic En signal between
wild-type and hth mutant embryos (data not shown), showing that the
repression activity of En is specifically affected in the hth
mutants.
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Although repression of slp was strongly reduced in hth mutants, it was not abolished. With longer inductions of hs-En expression, slp was more effectively repressed, again consistent with a residual activity in hth mutants (data not shown). A possible explanation for this residual activity is that the hthP2 allele is not a complete null. Alternatively, En might retain some repression activity even in the absence of Hth.
We used the same approach to test whether En activity in embryos requires Exd, using a null allele of exd. As shown in Fig. 1F-H, complete removal of both maternal and zygotic exd function caused a dramatic drop in En repression activity on the slp gene. However, as with hth, there appeared to be residual activity even in the absence of exd function. Thus, although En retains a residual repression activity in vivo without Hth and Exd, its effectiveness is severely reduced in the absence of either.
Repression of both wingless and slp by En is
facilitated by exd
To examine further the requirements for exd and hth by
En, we expressed En ectopically using the Gal4-UAS system
(Brand and Perrimon, 1993), in
a pattern that partially overlaps with the expression of two En target genes,
wg and slp. This patterned ectopic expression provides an
internal control within individual embryos of unrepressed target gene
expression, as well as avoiding the potential complicating effects of heat
shock. In this system, a transgene (the driver) expresses the Gal4 activator
protein in a pattern, and this drives the expression of a responding
transgene, in this case UAS-en. Using the prd-Gal4 driver
[made by L. Fasano and C. Desplan (see
Yoffe et al., 1995
)],
UAS-en is activated in a striped pattern
(Fig. 2B) that covers every
other stripe of both wg and slp in the segmented portion of
the embryo (wg is expressed within the slp domain, just
anterior to each normal en stripe). The effects of this ectopic En
expression were assayed in wild-type and exd mutant embryos. The
effects on wg expression are shown in
Fig. 2. Alternate wg
stripes (in even-numbered parasegments) were repressed completely in wild-type
embryos (Fig. 2D), whereas
repression of these stripes was less complete in embryos that lacked a
maternal supply of Exd protein and also had a reduced zygotic gene dose, and
appeared to be delayed relative to the wild-type controls
(Fig. 2F, and data not shown;
notice that some expression remains, particularly in parasegments 0, 2 and 4).
In embryos that completely lack exd function (both maternally and
zygotically), the situation is complicated by the fact that continued
wg expression requires exd. The weakening of wg
stripes in exd null embryos might be secondary to the loss of
en expression, because en is necessary to maintain
hedgehog and wg transcription
(Heemskerk et al., 1991
;
Ingham, 1993
). Thus, even
without ectopic En expression, wg expression is weaker, particularly
in odd-numbered parasegments such as 3 and 5
(Fig. 2G,I). Nevertheless,
against this `background' of weakened odd-numbered wg stripes, the
ability of prd-Gal4-driven En (expressed only in even-numbered
parasegments) to repress wg was significantly reduced. Although this
effect was also seen in more-posterior parasegments, the effect was noticeably
stronger anterior to the abdomen (particularly in parasegments 0, 2 and 4),
where there appeared to be very little repression of wg compared with
exd mutant embryos in which En was not ectopically expressed
(Fig. 2G-J).
In similar collections of embryos, we examined the consequences of loss of exd function on the ability of En to repress slp. As with wg, alternate slp stripes were strongly repressed by prd-Gal4-driven En (Fig. 3A,B). Simultaneously eliminating the maternal contribution and reducing the zygotic dose of exd+ caused a delay in repression and an ultimately less-complete reduction of slp expression (Fig. 3E,F). In embryos completely lacking both maternal and zygotic exd function, repression activity was strongly reduced (Fig. 3G,H), consistent with the results involving ubiquitously expressed En (Fig. 1). As with repression of wg, the repression of slp by En was more dependent on exd function in the gnathal-thoracic region than in the abdomen (Fig. 3H, parasegments 0, 2 and 4 vs 6, 8, 10 and 12). However, even in the abdomen, repression by En was significantly reduced in the absence of exd (Fig. 3G,H vs A,B).
Activity of En in pattern formation is dependent on both exd
and hth
To test the significance of the changes in the specific En target genes
described above for the overall ability of En to regulate its target genes, we
examined the extent to which reduced hth and exd function
affected the ability of ectopically expressed En to alter the pattern of
structures produced at the end of embryogenesis in the external larval
cuticle. First, in hth mutant embryos, we examined the severity of
cuticle defects produced by hs-En. As
Table 1 shows, the ability of
hs-En to cause severe cuticle defects was reduced in hth
heterozygotes and homozygotes, suggesting that the observed reductions in En
repression activity on the specific target genes wg and slp
accurately reflects its overall ability to regulate the target genes
responsible for cuticle pattern.
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We also quantified the severity of cuticle defects caused by ectopically patterned En expression and compared the results between the exd+/+ and exd+/- populations, which are both wild type in the absence of hs-En. The analysis (Fig. 4) showed that, in the embryo, the reduction in exd activity caused by simultaneously removing maternal exd function and reducing the zygotic gene dose had a significant effect on the ability of prd-Gal4-driven En expression to disrupt the proper development of abdominal cuticular structures. This confirms the developmental impact of the changes in target gene expression characterized above. These results also confirm that En has a significant requirement for exd in the abdomen, as well as in more anterior regions.
En interacts directly with both Exd and Hth
As a first step in determining the mechanisms whereby exd and
hth contribute to repression by En in vivo, we examined the
possibility of direct interaction. Previous studies had shown that En can bind
co-operatively with Exd in vitro to artificial DNA sites
(Peltenburg and Murre, 1996).
We tested whether a direct En-Exd interaction could also occur in other
contexts, using yeast two-hybrid and in vitro assays. We also tested whether
En could interact similarly with Hth. Fig.
5A shows the results of two-hybrid assays in yeast. En appeared to
interact robustly with Exd in this system, because the signal strength
observed with both isolated colonies (data not shown) and colony streaks
(Fig. 5A) was consistently
higher than that seen with some of our positive controls, including the
functionally important interaction between En and Groucho (data not shown)
(Tolkunova et al., 1998
). This
signal was also comparable to that seen with Exd and the mouse homolog of Hth,
Meis1 (Fig. 5A; Hth fused with
the Gal4 DNA-binding domain gave a high background signal, so that parallel
results using Hth with Exd were uninformative). En also gave a somewhat
weaker, but apparently specific, signal in combination with either Hth or
Meis1 (Fig. 5A).
In vitro, En also interacts specifically with both Exd and Meis1 (Fig. 5B). Here, En fused with GST, but not GST alone, effectively pulls down either Exd or Meis1. Meis1 was used in these studies because of the high level of non-specific interaction observed with in-vitro-translated Hth, perhaps owing to the heterologous nature of the translation system. In this system, it is unlikely that the interactions are due to co-operative binding to DNA, and we interpret these results to mean that these interactions can occur in solution. Furthermore, Meis1 appears to interact more strongly with En in the presence of Exd, suggesting that the three proteins form a co-complex.
We tested whether these molecular interactions can also occur in cultured
Drosophila cells (S2 cells). We transfected these cells with a Hth
expression plasmid, which induces nuclear localization of endogenously
expressed Exd (Rieckhof et al.,
1997), either alone or with expression plasmids for En and for a
tagged form of Exd (His6Exd, see Materials and Methods). In nuclear
extracts from these cultures, anti-Hth antiserum specifically precipitated
both En and endogenous Exd, as well as the tagged form of Exd
(Fig. 5C). Conversely,
anti-His6 antibodies specifically precipitated both En and Hth
(Fig. 5C). This ability of
either Hth or His6Exd to mediate precipitation of En occurs at a
salt concentration of 150 mM and with 0.2% Triton (see Materials and Methods),
suggesting that the complexes involved are reasonably stable. However, the
interaction of Hth with Exd, which is known to be functionally important,
might be somewhat more stable, because the background seen under conditions
needed to generate a readily detectable signal is somewhat lower between Hth
and endogenous Exd, and between His6Exd and transfected Hth, than
that seen with En (notice the slight background visible in the `
antibody' lane at the positions of En and tag-Exd in
Fig. 5C, and the lack of any
background at the positions of endogenous Exd and Hth). Although the
interactions seen with En might be weaker than those between Exd and Hth, they
are nonetheless strong enough to suggest the direct involvement of Exd and Hth
in repression by En in vivo.
Hth and Exd augment repression by En in cultured cells
As a first step in assessing the influence of the physical interactions
identified above on the transcriptional activity of En, we used a previously
constructed co-operative binding site for En and Exd
(Peltenburg and Murre, 1996)
as a target site in transfection assays. Repression by En has been extensively
studied in Drosophila S2 cells, which have been shown to express Exd,
but not Hth, constitutively (Rieckhof et
al., 1997
). Co-operative binding sites were inserted into a
reporter vector previously shown to be unresponsive to En in the absence of
inserted sites (Jaynes and O'Farrell,
1991
) and their responses to En and to En in combination with Hth
were measured. Consistent with previous results, En was able to repress this
artificial target gene only when it contained the inserted sites and, without
these sites, neither Hth alone nor En plus Hth caused any repression
(Fig. 6A). Importantly, with
the binding-site-containing reporter, the presence of Hth significantly
increased the effectiveness of repression by En, either on the basal
expression level of the target gene (Fig.
6A) or, in an established assay for active repression, when this
gene was activated from separate activator binding sites
(Fig. 6B). Because Hth has been
shown to induce the nuclear localization of endogenously expressed Exd in
these cells (Abu-Shaar et al.,
1999
), this might be due to an increased nuclear concentration of
En-Exd complexes, to a Hth-En interaction or to a combination of the two.
In order to test whether the increase in repression by En caused by
expression of Hth was mediated by cooperative binding between En and Exd, we
tested mutated versions of the cooperative binding site. A two-nucleotide
change in an Exd core consensus binding sequence (TGAT) within the
30-nucleotide oligonucleotide has previously been shown to abolish
co-operative binding by En and Exd (van Dijk, 1994). As shown in
Fig. 6, this Exd-site-mutated
target gene still responded to En, but the increased repression caused by Hth
was largely absent. Although there was no apparent increase in repression of
basal transcription caused by Hth with this mutated target gene, some residual
increase in repression of activated transcription appeared to persist. This
was true even when the other core Exd consensus binding sequences (ATCA)
within the oligonucleotide were also mutated (data not shown). Thus,
eliminating co-operative binding between Exd and En significantly reduced the
cooperative repression caused by Hth expression, suggesting that this effect
of Hth is mediated at least in part by co-operative binding between Exd and
En, whereas a secondary effect of Hth expression on repression of activated
transcription might be independent of such co-operative binding. Consistent
with previous results (Jaynes and
O'Farrell, 1991), when the En core consensus sequence ATTA was
mutated to AGGA, repression by En, or by En plus Hth, was eliminated (data not
shown).
To test the influence of Exd more directly, we turned to RNA interference
(Clemens et al., 2000) to
reduce endogenous Exd levels in S2 cells. As shown in
Fig. 6C, treatment of S2 cells
with Exd dsRNA reduced En-mediated repression from the co-operative binding
sites significantly, relative to non-specific dsRNA. This effect was not seen
at higher concentrations of En, possibly owing to saturating levels of En.
However, the increase in repression by Hth was clearly reduced by Exd dsRNA at
both high and low En concentrations, suggesting that Hth and Exd cooperate to
increase the repression activity of En.
Although these results might be accounted for by a combination of Hth-induced nuclear localization of Exd and co-operative binding by Exd and En to the target gene, the observed ability of En to form complexes with both Hth and Exd suggest the simple model that repression by En alone is less effective than repression by a trimolecular complex of En, Exd and Hth.
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DISCUSSION |
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Exd and Hth form complexes with Engrailed
En can form complexes with both Exd and Hth (as well as its mammalian
homolog Meis1) in vitro, in yeast and in cultured cells
(Fig. 5). Interestingly, rather
than the two-way interactions being mutually exclusive, En appears to be able
to interact simultaneously with Exd and Meis1 in vitro
(Fig. 5B). In extracts from
transfected cultures, En is specifically precipitated by antisera against both
Exd and Hth (Fig. 5C).
Therefore, En might interact with these cofactors similarly to Hox proteins,
which have been shown to form such three-way complexes
(Ferretti et al., 2000;
Jacobs et al., 1999
;
Ryoo et al., 1999
).
In cultured Drosophila cells, Exd and Hth cooperate with En to
repress transcription. Using a co-operative binding site for Exd and En
(Peltenburg and Murre, 1996)
to construct an En-responsive target gene, we found that both Exd and Hth are
required for full repression activity (Fig.
6). When a mutation was introduced into an Exd consensus binding
sequence that eliminates co-operative binding, co-operative repression was
largely eliminated, whereas mutating the En consensus binding sequence
eliminated repression. This, along with the fact that RNA interference
directed against Exd mRNA also largely eliminated co-operative repression
(Fig. 6C), suggests that a
complex containing Exd and En is responsible for the co-operative repression
caused by coexpression of Hth and En (Exd is constitutively expressed in these
cells). Because Hth regulates the nuclear localization of Exd, it can allow
Exd-En repression complexes to form in the nucleus. In addition, the observed
molecular interactions suggest that the fully active repression complex might
include all three proteins.
Exd cooperate s with En to repress target genes and to pattern
embryos
Loss of exd function has been shown to result in a loss of
en expression at later embryonic stages
(Rieckhof et al., 1997).
Because en function is required to maintain its own expression
(Heemskerk et al., 1991
), the
loss of en expression could be a downstream effect of a loss of
en function, or it could be due to some other consequence of the lack
of exd. This ambiguity concerning the role of exd in
en function led us to investigate whether the activities of
ectopically expressed En (Heemskerk et
al., 1991
) are dependent on exd function. We expressed En
ectopically in two ways: from a heat-shock promoter and using a patterned Gal4
`driver' transgene. An advantage of the former approach is that one can often
distinguish between immediate and secondary downstream effects based on how
rapidly they occur following heat induction. Advantages of the second approach
include having normal and altered expression in parts of the same embryo,
providing a rigorous internal control. Both of these approaches led to similar
conclusions (Figs
1,2,3,4),
that exd function is important for the repression by En of its direct
target gene slp, that wg also shows a strong dependence on
exd function for its repression by En and that the ability of En to
alter the pattern of embryonic cuticles is sensitive to the gene dosage of
exd. Further, in each set of experiments, the observed dependence of
repression on exd was accompanied by a residual repression activity
when exd function was removed both maternally and zygotically. This
residual exd-independent repression activity might be due to the
ability of En to bind to target sites independently of exd but with a
reduced affinity, or it could be accounted for by the existence of two classes
of binding sites, one exd dependent and the other exd
independent. This possibility is paralleled by the relationship of Exd with
Ubx, which has been shown to function either co-operatively with Exd or alone
on multiple binding sites in target genes
(Galant et al., 2002
).
Alternatively, exd might be exerting an indirect effect on repression
by En. However, because Exd forms complexes with En in yeast and in vitro, and
because it appears to facilitate repression by En directly in cultured cells,
it seems likely that the dependence of En on exd function in vivo is
due at least in part to the direct action of En-Exd complexes. Confirmation of
this model will require the analysis of specific regulatory sites, which have
not yet been identified, in target genes such as slp. If this model
is correct then our results suggest that the repression activity of Exd-En
complexes might come exclusively from En repression domains
(Han and Manley, 1993
;
Jaynes and O'Farrell, 1991
;
Jimenez et al., 1997
;
Smith and Jaynes, 1996
;
Tolkunova et al., 1998
),
because Exd has been shown to act as a cofactor in the activation of target
genes in vivo in conjunction with Hox proteins (reviewed in
Mann and Chan, 1996
;
Grieder et al., 1997
;
Inbal et al., 2001
;
Liu et al., 2001
;
Pinsonneault et al., 1997
;
Ryoo et al., 1999
).
The effects of eliminating exd function on repression by En appear
to be different in the abdomen and the more-anterior regions (Figs
2,
3), in that En is less
dependent on exd in the abdomen (parasegments 6-12). Similar results
have recently been obtained by Alexandre and Vincent
(Alexandre and Vincent, 2003),
as described in their accompanying paper. One possible explanation is that
hth can provide the observed exd-independent activity.
However, in exd mutants, Hth levels are reduced, probably because Hth
protein is less stable without Exd
(Abu-Shaar et al., 1999
).
Nevertheless, our data are consistent with the possibility that, on their own,
either Hth or Exd might provide partial cofactor activity, whereas both
together might be required for full activity. The latter possibility is
suggested by our observation that maximal repression activity in S2 cells
requires all three gene products.
An additional possibility to account for the residual exd- and
hth-independent repression activity of En in the abdomen is that
other cofactors assist En in binding to its target genes in the abdomen. If
there are other cofactors at work, it is likely that their activity (or
expression) is dependent, either directly or indirectly, on the Hox genes
Ubx and abd-A, because these genes are responsible for all
known aspects of differential segment identity in this region of the embryo.
This expectation has been directly confirmed by Alexandre and Vincent
(Alexandre and Vincent,
2003).
It is noteworthy that the difference in the dependence of En on
exd in the abdomen versus the thorax is seen only after stage 9 (for
example, it is not seen in Fig.
1), when the levels of Hth, and the consequent nuclear
concentration of Exd, have declined in the abdomen
(Rieckhof et al., 1997). Thus,
the dependence of En on exd parallels the nuclear concentration of
Exd, and might reflect an evolutionary adaptation to the changing levels of
Exd in different regions of the embryo.
Requirements for hth and exd in En activity are
similar in vivo
Hth has been shown to act in part through its facilitation of the nuclear
localization of Exd, and strong hth and exd mutants have
very similar phenotypes (Rieckhof et al.,
1997). Although Hth can also interact with En independently of Exd
(Fig. 5), transfection assays
in cultured cells suggest that Hth might depend entirely on Exd for its
ability to increase repression by En, at least from artificial En-Exd
co-operative binding sites (Fig.
6C). Because Hth forms complexes with En in these cells
(Fig. 5C), in addition to
increasing its repression activity (Fig.
6A,B), a simple model is that maximal repression activity is due
to complexes containing En, Exd and Hth. However, we cannot rule out the
possibility that Hth acts solely by making Exd available to interact with En
on target sites, through its ability to bring Exd into the nucleus.
We tested whether the repression activity of ectopically expressed En in vivo is dependent on hth function, using assays similar to those used for exd (Fig. 1, Table 1). In each case, we observed a close similarity to results with exd mutants. En activity showed a strong dependence on hth function, although residual activity remained in hth mutants. In addition, En activity showed a sensitivity to the hth gene dose. All of these results are consistent with the effects of Hth being exerted through its effect on Exd nuclear localization, provided that the nuclear targeting of Exd is necessary for its ability to function with En. However, as noted above, Hth might also increase the effectiveness of En repression directly, by forming complexes with En and/or as part of En-Exd complexes. A detailed analysis of a number of in vivo target sites will be necessary to distinguish among these possibilities.
New exd and hth functions
Exd and Hth are essential to the correct regulation of target genes by the
homeodomain proteins of the Hox clusters (reviewed in
Mann and Affolter, 1998).
However, their functional interactions have not previously been shown to
extend beyond the highly restricted subset of homeodomain proteins that are
found within the Hox clusters (the Antp, Abd-B and Labial classes). The
identification of functional interactions with En suggests that exd
and hth might provide functional specificity in conjunction with
other non-Hox-class homeodomain proteins.
The identification of slp as a direct target gene of En has
implications for the mechanism by which En helps to maintain the activity of
its own and other genes, including hedgehog, within its domains of
expression, the posterior compartments. The slp locus produces two
closely related, coordinately regulated gene products (Slp1 and Slp2), which
have essentially indistinguishable functions
(Cadigan et al., 1994;
Grossniklaus et al., 1992
).
They are forkhead-domain transcription factors
(Grossniklaus et al., 1992
)
that repress en expression
(Cadigan et al., 1994
), and
both contain a conserved motif (homology region II)
(Grossniklaus et al., 1992
)
that is similar to the Groucho-binding domain of En
(Tolkunova et al., 1998
). Slp1
has also been shown to bind the Groucho co-repressor in vitro
(Kobayashi et al., 2001
),
suggesting that it is a repressor and therefore that its action on the
en gene is likely to be direct. Thus, the mechanism of en
autoregulation, as well as the ability of En to activate other target genes,
is likely to be due, at least in part, to an indirect effect of repression of
slp expression. Similar conclusions have been reached by Alexandre
and Vincent (Alexandre and Vincent,
2003
), as described in the accompanying paper. In addition, En
might activate target genes indirectly by repressing other repressors that are
also normally excluded from its expression domain, such as Odd-skipped
(Mullen and DiNardo, 1995
;
Saulier-Le Drean et al., 1998
)
and the repressor form of Cubitus interruptus
(Eaton and Kornberg,
1990
).
Although there have been previous suggestions that Exd and Hth might
participate in active repression as well as activation complexes
(Abu-Shaar and Mann, 1998;
Abzhanov et al., 2001
;
Manak et al., 1994
;
White et al., 2000
), most of
the well-characterized direct Exd-Hth-Hox target genes are activated in an
exd- or hth-dependent fashion
(Manak et al., 1994
;
Pinsonneault et al., 1997
;
Ryoo and Mann, 1999
;
Ryoo et al., 1999
). In fact,
these observations raised the question of whether Exd and Hth might be
dedicated to gene activation (Pinsonneault
et al., 1997
). Recently, Hth and Exd have been shown to act
directly with Ubx to repress the Hox target gene Distalless in the
Drosophila abdomen (Gebelein et
al., 2002
). The partnership with En in repression further argues
that these cofactors can increase the target site discrimination of
homeodomain proteins without restricting the resulting transcriptional
activity to activation alone. Based on these results, we suggest that Hth and
Exd increase the target-site discrimination of several classes of homeodomain
proteins and that they do so without defining the transcriptional activity of
the resulting protein complex.
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ACKNOWLEDGMENTS |
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