1 Section in Cell and Developmental Biology, Division of Biology, University of
California, San Diego, La Jolla, CA 92093, USA
2 Howard Hughes Medical Institute, Laboratory of Genetics, University of
Wisconsin-Madison, Madison, WI 53706, USA
* Author for correspondence (e-mail: mcginnis{at}biomail.ucsd.edu)
Accepted 28 September 2005
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Ultrabithorax, Drosophila, Hox, Transcriptional activation, Transcriptional repression, Sex combs reduced
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
In the visceral mesoderm (VM), Ubx activates the transcription of the
decapentaplegic (dpp) gene in parasegment 7
(Capovilla and Botas, 1998;
Manak et al., 1995
;
Muller et al., 1989
;
Sun et al., 1995
;
Tremml and Bienz, 1989
), where
dpp is required for the formation of the second midgut constriction
(Immerglück et al., 1990
;
Reuter et al., 1990
). In the
epidermis of the embryonic trunk, Ubx activation function is required for the
maintenance of the transcription of teashirt (tsh), a
homeotic gene that acts in concert with trunk Hox genes to promote trunk
identity (Fasano et al., 1991
;
McCormick et al., 1995
;
Roder and Kerridge, 1992
). Ubx
provides specific segmental identity to parasegment 6, in part by repressing
the transcription of another Hox gene, Antennapedia (Antp)
(Carroll et al., 1986
;
Hafen et al., 1984
;
Saffman and Krasnow, 1994
). In
the abdominal ventral epidermis, the Ubx and Abd-A Hox proteins prevent the
formation of embryonic limbs by directly repressing the transcription of the
Distal-less (Dll) appendage-promoting gene
(Vachon et al., 1992
).
Ubx homologs from some evolutionarily distant species can appropriately
regulate Drosophila Ubx target genes in embryonic assays, suggesting
evolutionarily conservation of activation and repression functions in these
proteins (Galant and Carroll,
2002; Grenier and Carroll,
2000
; Ronshaugen et al.,
2002
). It is therefore of great interest from an evolutionary
point of view to understand which regions in Ubx contribute to its activation
and repression functions, and whether they are conserved among other Hox
proteins.
Many studies have focused on mapping domains required for Ubx limb
repression functions in embryos, which is largely due to the ability of Ubx to
transcriptionally repress Dll
(Vachon et al., 1992). Some of
these studies have come to different conclusions. For example, a recent study
has provided evidence that the domain encoded in the optional exon, present in
Ubx isoforms Ia and Ib, but absent from the isoform IVa, is required for the
repression of larval limbs (Keilin's organs) and Dll transcription
(Gebelein et al., 2002
).
However, three earlier studies found that Ubx isoform IVa was as effective, or
nearly as effective, as the Ib isoform at repressing limbs
(Busturia et al., 1990
;
Mann and Hogness, 1990
;
Subramaniam et al., 1994
).
In order to address such inconsistencies, and learn more about Ubx
activation and repression functions, we have performed quantitative assays of
Ubx function, and find that the repression activity of Ubx in embryos is
highly concentration dependent. Using this knowledge and deletion mutants, we
have mapped domains required for the repression and activation functions of
Ubx protein. A domain required for transcriptional activation, which includes
a variant of the Ser-Ser-Tyr-Phe (SSYF) amino acid motif that is
evolutionarily conserved in many Hox proteins, maps to the N-terminal 19 amino
acids. Although the YPWM region upstream of the homeodomain is required for
Ubx to repress Dll with normal cooperativity, no single deletion
abolishes the Ubx repression function. Instead, in combination with other
findings (Hittinger et al.,
2005), our data suggest that the Ubx protein contains multiple
regions that contribute additively to its repression function on embryonic
targets.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Immunostaining and quantitation of the protein expression levels.
Experimental and control embryos were collected and processed
simultaneously for immunostaining as previously described
(McGinnis et al., 1998),
except that Western Blocking Reagent (Roche) was used for blocking. Ubx was
detected with FP3.38 antibody (White and
Wilcox, 1984
); HA-tagged proteins were detected with rat anti-HA
antibody (Roche). Embryos were mounted in FluoroGuard Antifade Reagent
(BioRad) and unsaturated images of ectodermal staining of early stage 11
embryos were taken using confocal microscope (Leica Microsystems), using
identical settings between experimental and control samples. Average levels of
pixel intensity were measured in the nascent limb field area in the transgenic
embryos and in the corresponding area of the first abdominal segment of the
wild-type control, using Leica Confocal software. After subtraction of the
background, which was measured in ventrolateral thorax of the same stage
wild-type embryos, the ratios between the experimentally induced protein
levels and endogenous Ubx protein levels were determined. Scr protein
concentration was determined similarly, using rabbit anti-Scr antibody; CrebA
protein was detected using rat anti-CrebA antibody (both gifts from D.
Andrew).
In situ hybridization and quantitation of the transcription levels
In situ hybridization was performed as described by Kosman et al.
(Kosman et al., 2004). The
Dll antisense probe was made from a 1.4 EcoRI cDNA fragment
(Cohen et al., 1989
), the
AntP1 probe was as described by Bermingham et al.
(Bermingham et al., 1990
), the
dpp probe was made from a 3.5 kb cDNA in pNB40 (a gift from E. Bier),
the tsh probe was produced from BSKSNotI-tsh plasmid
(Fasano et al., 1991
), the
wg probe was as described by Cohen
(Cohen, 1990
) and the
fkh probe was produced from a 1.5 kb pBst-fkh plasmid.
Quantitation of the transcriptional repression of Dll and activation
of dpp was performed using the histogram function of Adobe Photoshop.
The background pixel intensity was measured in the same embryo, in the areas
adjacent to the signal, and subtracted from the average signal value.
Curve fitting and analysis
The data points of Dll transcriptional repression versus Ubx
concentration were processed using GraphPad Prism 4 Software as follows: Ubx
concentration values were transformed to logarithmic values, a non-linear
regression analysis option was chosen and a sigmoidal dose-response (variable
slope, Y=Top/(1+10(LogEC50-X)^HillSlope) curve was fitted to the
data. The goodness of the fit of the resulting curves, measured as the
coefficient of determination (R2), was 0.97 for wild-type Ubx and
0.96 for UbxYPWM.
Sequence alignments
Sequence alignments and processing were performed using ClustalW and
Boxshade 3.21 programs available at the Swiss node of EMBnet
(http://www.ch.embnet.org).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The relationship between Ubx protein concentration and larval limb elimination is plotted in Fig. 1A. From 0-20% of endogenous protein levels, ectopic Ubx did not eliminate Keilin's organs (Fig. 1A, black curve). However, in the interval where ectopic Ubx increased from 20% to 70% of endogenous Ubx protein levels, there was a switch to a limbless state. The Keilin's organs developing in the presence of low Ubx concentration are not unaffected: even at 20% of the endogenous concentration, Ubx eliminates half of the sensory hairs of these rudimentary limbs (Fig. 1A, red curve). At 50% of the endogenous Ubx level, about 80% of the sensory hairs are eliminated and most Keilin's organs consist of the organ's base with a single sensory hair (Fig. 1A; data not shown).
We next tested whether a similar concentration-dependent relationship
existed between Ubx protein concentration and Dll transcripts in the
embryonic limb fields. In stage 11 embryos, Dll is transcriptionally
activated in the limb primordia of the three thoracic segments
(Fig. 1G). These are the cells
that will give rise to the Keilin's organs, and Dll is required for
the formation of both the base and the sensory hairs of the organ
(Cohen et al., 1991). The
repression of Dll transcription by ectopic Ubx is highly
concentration dependent, and follows closely the dose-response curve for the
repression of sensory hairs (Fig.
1B). The curve that best fits the data points for the Ubx protein
concentration-Dll transcript repression response has a sigmoidal
shape characteristic of cooperative biological regulatory systems in which
small changes in concentration trigger an abrupt transition from one state to
another (Johnson et al., 1981
;
Perutz, 1989
).
|
Protein domains required for repression of thoracic limbs
With the above concentration dependence in mind, we tested the larval limb
repression functions of eight mutant Ubx proteins (tagged with HA) containing
small deletions in regions N-terminal of the homeodomain
(Fig. 2A). We placed the
borders of our deletions between evolutionarily conserved regions of the Ubx
protein sequence (Fig. 2A, see
Fig. S1 in the supplementary material). These deletions span over 275 amino
acids, covering approximately three-quarters of the Ubx protein. Multiple
transgenic lines carrying the mutated forms of UbxIa protein under the control
of UAS regulatory sequence were generated and crossed to flies carrying
armadillo-Gal4 drivers. Expression levels of the mutant proteins were
compared either directly to the level of the endogenous Ubx in the first
abdominal segment (A1) of wild-type embryos, or indirectly, by comparison with
a line which ectopically expresses HA-tagged wild-type Ubx at an average of
76% of endogenous levels, and provides 100% limb repression
(Fig. 2B). All of the deletion
mutants produced proteins that were almost exclusively localized in nuclei,
with the exception of Ubx2-19, which was slightly defective in this
regard. It showed a ratio of nucleus to cytoplasmic protein staining of 3 to
1, so the expression values we report for this mutant have had cytoplasmic
levels subtracted.
|
The Ubx deletion mutant with the most severe defect in limb repression
lacks the YPWM motif and a few adjacent amino acids (Ubx234-251). When
produced at the levels of endogenous Ubx, the Ubx
YPWM mutant repressed
only 65% of larval limbs (Fig.
3A,B). Even when expressed at 170% of the endogenous
concentration, this mutant protein did not completely repress limbs (83%
repression, Fig. 3B). The
concentration dependence of the Ubx
YPWM-induced limb repression was
also notably less steep than is observed for wild-type Ubx
(Fig. 3B).
|
The five other N-terminal deletion mutants were potent repressors of larval
limbs when expressed at endogenous Ubx levels
(Fig. 3A). They also showed
steep concentration dependence curves, although at lower concentrations none
repressed limbs quite as effectively as wild type Ubx (data not shown).
Although previous research had suggested an important role in limb repression
for the alternatively spliced linker region absent in Ubx IVa
(Gebelein et al., 2002), our
data for Ubx
252-280 agree with earlier results suggesting that this
region is not essential for limb repression
(Busturia et al., 1990
;
Mann and Hogness, 1990
;
Subramaniam et al., 1994
).
The importance of the C-terminal region of Ubx, not covered in our deletion
series, was quantitatively assayed by Ronshaugen et al.
(Ronshaugen, 2002). In that
study, a Ubx mutant without the conserved C-terminal QA motif was expressed at
80% of the levels of wild-type Ubx, and was found to be 20% less
effective at limb repression than wild-type Ubx. We did not pursue a more
detailed quantitative analysis of the C-terminal region using ectopic
expression assays, as other studies
(Hittinger et al., 2005
) used
allelic replacement to generate a Ubx C-terminal deletion mutant, and found
that limb repression activity of the mutant protein was only slightly reduced
in embryos.
UbxYPWM mutant is an ineffective repressor of Dll and Antp
We next tested the function of the most defective Ubx deletion mutant,
UbxYPWM, on two known repression targets of Ubx protein, Dll
and the Antp P1 promoter
(Bermingham et al., 1990
;
Vachon et al., 1992
).
Wild-type Ubx and Ubx
YPWM mutant proteins were expressed at similar
levels (wild-type Ubx 32±5%, Ubx
YPWM 40±4%), and assayed
for their ability to repress Dll and Antp P1 transcripts.
Under these conditions, ectopic wild-type Ubx represses
85% of
Dll transcript levels in the anterior compartment of the limb field
(Fig. 4C). The Ubx
YPWM
deletion mutant is a less effective repressor of Dll transcription,
repressing 57% of Dll transcript levels in the anterior compartment
(Fig. 4E). The Ubx
20-61
protein exhibited a similar defect in Dll transcriptional repression
(not shown). The other Ubx deletion mutants, including Ubx
2-19 (which
we show later is required for Ubx transactivation function) repressed
Dll transcription to similar levels as wild-type Ubx, consistent with
their strong repression of larval limbs.
|
The Ubx YPWM deletion mutant has decreased repression cooperativity
At wild-type expression levels, the YPWM deletion mutant retains
significant limb repression ability, but the curve relating its protein
concentration to limb repression is much shallower than for wild-type Ubx. To
test whether a similar relationship exists between UbxYPWM protein
concentration and Dll repression, we quantified the repression of
Dll transcription in the anterior compartments of the thoracic
segments of embryos from the transgenic lines expressing a range of ectopic
Ubx
YPWM concentrations. Fig.
4G presents these data as a dose-response plot, where Dll
transcriptional repression is plotted as a function of the log [10] of ectopic
protein concentration. For wild-type Ubx, in black, the curve that best fits
the data is a steeply rising sigmoid curve. The steepness of the curve can be
measured by the Hill slope, which also provides a rough measure of the
cooperativity of the repression system. A Hill slope of 1 indicates that the
repression system lacks cooperativity, while a Hill slope of more than 1
indicates positive cooperativity. The Hill slope for the wild-type Ubx
repression curve is 4.9±2.2 (±two standard errors of the mean).
By contrast, the YPWM deletion dose-response curve is much shallower, with a
Hill slope of 1.7±0.8. The Hill slopes for wild-type Ubx and
Ubx
YPWM curves are statistically significantly different (F test,
P=0.006), indicating that the repression cooperativity of the YPWM
deletion mutant on Dll is reduced when compared with wild-type
Ubx.
A conserved region required for activation function of Ubx protein
In order to identify the regions required for the transcriptional
activation function of Ubx, we assayed the function of the Ubx deletion
mutants on two known activation targets of the endogenous Ubx protein, the
genes dpp and tsh
(Capovilla and Botas, 1998;
McCormick et al., 1995
;
Roder and Kerridge, 1992
;
Sun et al., 1995
).
Ectopic expression of wild-type Ubx at 100% of endogenous levels induces
robust activation of dpp transcription in the visceral mesoderm
anterior to parasegment 7, as well as in two weaker stripes posterior to
parasegment 7 (Capovilla et al.,
1994) (Fig. 5B).
Although the ectopic expression of the Ubx
YPWM mutant in the visceral
mesoderm was at only 60% of endogenous levels, it activated ectopic
dpp transcription in a pattern and amount indistinguishable from
wild-type Ubx (Fig. 5E,F). The
Ubx
20-61 mutant was a poorer dpp activator than wild type,
inducing no expression posterior to parasegment 7, and 30% lower levels in
parasegments 5 and 6 (Fig.
5D,F). This and previous data indicates that Ubx
20-61 is
partially defective in both repression and activation. We conclude that the
Ubx
20-61 mutant has a general defect in gene regulation, perhaps owing
to a change in protein structure caused by the deletion.
All but one of the other deletion mutants, including a deletion mutant
lacking the conserved C-terminal QA domain
(Ronshaugen et al., 2002),
produced dpp activation levels similar to wild-type Ubx (data not
shown). The notable exception to this was Ubx
2-19, which barely
activated dpp above background levels in parasegments 5 and 6
(Fig. 5C,F). Moreover, the
Ubx
2-19 mutant also repressed transcription of dpp in
parasegments 4 and 7 to barely detectable levels (compare
Fig. 5A with 5C). We concluded
that Ubx
2-19 was a defective activator of dpp transcripts, and
that the deletion of the Ubx 2-19 region converts it from an activator to a
repressor of dpp.
|
|
The conserved N-terminal region is required for Scr activation function
To test whether the N-terminal region of Hox proteins contains an
evolutionarily conserved activation domain, we assayed the function of this
region in another Hox protein, Sex combs reduced (Scr). The N terminus of
insect Scr proteins also contains an extremely well-conserved region
(Fig. 7A) with a significant
degree of sequence similarity to the N termini of Ubx and many other Hox
proteins (Fig. 7J). To
investigate the function of this region, we deleted 17 amino acids, starting
with the conserved SSYQFVN sequence (Fig.
7A). Multiple transgenic lines carrying wild-type Scr or its
N-terminal deletion mutant (ScrSSY) under UAS regulatory element
control were generated and crossed to the armadillo-Gal4 driver.
Expression levels of ectopic wild-type Scr and Scr
SSY were tested, and
lines were selected that ectopically expressed the proteins in the ventral
head at levels approximately equal to those of the endogenous Scr protein in
ventral parasegment 2 (Fig.
7B).
In wild-type embryos, Scr is required for the formation of salivary glands
in ventral parasegment 2 (Andrew et al.,
2000; Panzer et al.,
1992
). It does so by activating a battery of genes, among them
genes for the transcription factors Fork head (Fkh)
(Panzer et al., 1992
) and
CrebA (Andrew et al., 1994
).
Both genes are ectopically activated by ectopic Scr protein, and fkh
is a direct activation target of Scr (Ryoo
and Mann, 1999
).
Ectopic wild-type Scr induced robust activation of fkh
transcription in parasegment 1 (Fig.
7D, arrow). Ectopic fkh transcription was also activated
in the ventral region of the mandibular segment
(Fig. 7D, asterisk) and in the
procephalon. Ectopic ScrSSY protein was a much weaker activator of
ectopic fkh transcription, activating it only in a few cells of
parasegment 1 and the procephalon (Fig.
7D,E).
The ScrSSY protein was also a defective activator of the
CrebA gene. Ectopic wild-type Scr induced abundant ectopic expression
of CrebA protein in parasegment 1 (Fig.
7G, arrow). In addition, patches of CrebA expression were
activated in the procephalon and the ventral head area. The Scr
SSY
deletion mutant induced only a small patch of ectopic CrebA expression in the
posterior portion of parasegment 1 (Fig.
7H, arrow), and ectopic activation was also reduced in the
procephalon and the ventral head (Fig.
7H).
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The most relevant previous work on Hox N-terminal function in
Drosophila embryos involved tests of mouse HoxA5 deletion mutants
(Zhao et al., 1996). The
authors found that multiple regions N-terminal to the homeodomain were
required for HoxA5 to activate a forkhead promoter-reporter gene. One
of the required regions included amino acid residues 2-39, and the authors
proposed this region might be required for activation function or co-factor
specificity. Similarity of Hox protein N-terminal sequences in
Drosophila and mammals has been long noted, and is a characteristic
of Hox proteins from a wide variety of animal species
(Martinez et al., 1997
;
Schughart et al., 1988
;
Zhao et al., 1996
). In both
mammal and Drosophila Hox proteins, the core conserved motif in this
N-terminal region is a Ser-Ser-Tyr-Phe (SSYF) amino acid sequence
(Fig. 7J).
We do not yet know the mechanism through which the Hox SSYF activation
domain operates: it may interact with DNA-binding transcription factors
dedicated to transcriptional activation or with co-activator protein complexes
(Glass et al., 1997). One
possible SSYF interactor is the histone acetyltransferase CBP (CREB-binding
protein) (Chan and La Thangue,
2001
). Mutations in the Drosophila CBP gene were found to
be dose-sensitive modifiers of Deformed and Ubx biological
function (Florence and McGinnis,
1998
). In addition, CBP was found to increase the transactivation
activity of human HOXB7 protein in breast cancer cells and to interact with
the N-terminal region of HOXB7 in GST pull-down assays, in a manner that
required the presence of the first 18 N-terminal amino acids of HOXB7
(Chariot et al., 1999b
). In
another study, mammalian CBP was shown to interact with the first 141
N-terminal amino acids of human HOXD4 in co-immunoprecipitation assays, and to
increase transactivation activity of HOXD4-PBX complexes on a synthetic
element containing five HOX/PBX sites in cultured human embryonic kidney cells
(Saleh et al., 2000
). Another
possibility is that the N terminus interacts with the I
B
protein, which binds to the N-terminal regions of human HOXB7
(Chariot et al., 1999a
), a
region of HOXB7 that is required for normal function in a murine
myelomonocytic cell line (Yaron et al.,
2001
).
A detailed analysis of Ubx domains required for transactivation function in
Drosophila cultured S2 cells, which are derived from embryonic
hemocytes (Armknecht et al.,
2005), was carried out recently by Tan et al.
(Tan et al., 2002
). In their
assays, the N-terminal 67 amino acid residues were not required for
Ubx-dependent transcriptional activation. The disparity between our results
and those from Tan et al. (Tan et al.,
2002
) might be explained by the different assay systems (cultured
S2 cells versus embryos), the different target elements, and/or the exact size
and extent of the deletion mutants that were tested.
Cooperativity in Ubx transcriptional repression function
Our results indicate that at least for its limb and Dll repression
functions, Ubx contributes to a cooperative on/off switch over a small
concentration range. When Dll repression is plotted as a function of
Ubx concentration, the best-fit curve has a Hill slope of 4.9±2.2.
These results suggest a highly cooperative assembly of a multiprotein
repression complex containing Ubx on Dll regulatory DNA. Although our
repression dose-response curves cannot be extrapolated into the number of
cooperative protein-protein interactions within a repression complex, they are
a surprisingly good fit to the model of Gebelein et al.
(Gebelein et al., 2004). In
this model, the Ubx-mediated repression of a Dll limb enhancer
requires at least five clustered DNA sites that cooperatively bind two
molecules of Ubx, Extradenticle (Exd) and Homothorax, while the fifth site
binds the Sloppy paired 1 protein
(Gebelein et al., 2004
). The
high sensitivity of Ubx phenotypes to concentration may explain why previous
experiments using ectopic expression of Ubx have come to different
conclusions, and illustrates why the validity of conclusions from ectopic
expression studies should be interpreted with caution, unless great care is
taken to achieve near-normal physiological levels.
Why is the Ubx repressive effect on Dll so concentration
sensitive? It is instructive to look at other biological systems with similar
concentration-dependent transcriptional switches. For example, the steep
concentration dependence of the lambda transcriptional repressor allows
prophages in E. coli cells to switch, at crucial levels of cellular
distress, from one stable state to another, lysogenic to lytic
(Johnson et al., 1981). For
Ubx, one likely reason for the highly concentration-dependent effects on
Dll expression and limb development is to ensure that all the cells
in a limb field are stably programmed to adopt either the limb state, or body
wall fate. At least in extant Drosophila, a mosaic appendage that
developed from a mixed field of limb and body wall cells would presumably be
little benefit to the animal that carried it, and thus selected against during
evolution.
Cooperative repression and the Ubx YPWM region
Tests of mutant Hox proteins in Drosophila and in mice have
demonstrated the importance of the YPWM motif for Hox function in vivo,
although both loss- and gain-of-function phenotypes were observed
(Chan et al., 1996;
Galant et al., 2002
;
Medina-Martinez and Ramirez-Solis,
2003
; Merabet et al.,
2003
; Remacle et al.,
2004
; Zhao et al.,
1996
). In vitro, the YPWM region has been shown to mediate Hox
interactions with the PBC family of homeodomain proteins
(Chang et al., 1995
;
Johnson et al., 1995
;
Knoepfler and Kamps, 1995
;
Neuteboom et al., 1995
;
Passner et al., 1999
;
Phelan et al., 1995
;
Piper et al., 1999
;
Shanmugam et al., 1997
). The
PBC proteins (Exd protein in Drosophila, Pbx proteins in mammals)
bind cooperatively with Hox proteins on composite DNA sites, and are important
co-factors in the regulation of many Hox target genes
(Featherstone, 2003
).
Galant et al. (Galant et al.,
2002) found that a Ubx protein with a YAAA substitution for YPWM
exhibited reduced cooperative binding with Exd on a consensus composite
Ubx-Exd DNA-binding site. Reduced affinity between Ubx
YPWM and Exd
might compromise the assembly of the entire repression complex proposed by
Gebelein et al. (Gebelein et al.,
2004
), resulting in an inefficient transcriptional repression of
Dll in the anterior segmental compartments.
Our in vivo results are also consistent with models in which the YPWM
region contributes in other ways to repression cooperativity. For example, the
YPWM region appears to influence Hox activation and repression functions in a
manner that is independent of its role in enhancing the affinity of Hox/PBC
protein complexes for binding sites (Chan
et al., 1996; Merabet et al.,
2003
). In vitro, Ubx is also known to bind cooperatively to DNA in
homomeric complexes (Beachy et al.,
1993
), and the YPWM motif might be required for the formation of
such complexes on Dll regulatory sequences.
No single deletion abolishes the Ubx repression function, although some
regions are required for robust repression. Hox protein repression function
appears to be quite complex. Our embryonic tests of the deletion mutants, and
the results of others (Hittinger et al.,
2005), suggest that Ubx contains multiple regions that additively
contribute to repression. In addition, previous studies
(Catron et al., 1995
;
Li et al., 1999
;
Zhang et al., 1996
) suggest
that the homeodomain also contributes directly to transcriptional repression
function in a manner that is independent of its DNA-binding function.
The Ubx YPWM region and transcriptional activation
The deletion of the Ubx YPWM region had little detectable effect on the
transcriptional activation of the dpp and tsh genes. As
exd genetic function is required for normal levels of dpp
and tsh activation in Ubx-expressing cells
(Chan et al., 1994;
McCormick et al., 1995
;
Rauskolb and Wieschaus, 1994
;
Sun et al., 1995
), this result
is difficult to reconcile with a simple model in which the YPWM motif is
required for Exd recruitment to activation target sites in dpp and
tsh enhancers. However, it is consistent with studies that tested the
effect of YPWM mutations on the activation abilities of the Labial and Abd-A
Hox proteins in embryos (Chan et al.,
1996
; Merabet et al.,
2003
). A YPWM to AAAA mutant of Labial was a more potent activator
than wild-type Labial protein of a sequence derived from the Hoxb1
autoregulatory region (Chan et al.,
1996
), whereas a YPWM-to-AAAA mutant of Abd-A converted this
protein from a repressor into an activator of dpp transcription
(Merabet et al., 2003
). In
addition, this YPWM mutation had no effect on the activation function of Abd-A
on wingless. The ability of Labial and Abd-A YPWM mutants to retain
their transactivation functions is correlated with their ability to bind Exd
in vitro in a YPWM-independent fashion
(Chan et al., 1996
;
Merabet et al., 2003
). The
YPWM-independent interactions between Hox proteins and Exd can be mediated by
Hox homeodomains and the C-terminal regions
(Li et al., 1999
;
Chan et al., 1996
).
As the Ubx-responsive elements from dpp and tsh loci
possess a mixture of Ubx monomer and Ubx-Exd heterodimer-binding sites
(Sun et al., 1995;
McCormick et al., 1995
),
possible reasons for the ability of the Ubx YMPM deletion mutant to activate
these downstream target genes are: (1) Hox activation of target genes often
involves a mixture of Exd-dependent and Exd-independent functions
(Pearson et al., 2005
); (2)
removal of the YPWM motif does not completely abolish Exd-Ubx binding
interactions (Galant et al.,
2002
); and (3) the YPWM apparently serves other functions besides
binding Exd in the context of developing embryos
(Chan et al., 1996
;
Merabet et al., 2003
).
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
Footnotes |
---|
Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/132/23/5271/DC1
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Akam, M. E. (1983). The location of Ultrabithorax transcripts in Drosophila tissue sections. EMBO J. 2,2075 -2084.[Medline]
Andrew, D. J., Horner, M. A., Petitt, M. G., Smolik, S. M. and Scott, M. P. (1994). Setting limits on homeotic gene function: restraint of Sex combs reduced activity by teashirt and other homeotic genes. EMBO J. 13,1132 -1144.[Abstract]
Andrew, D. J., Henderson, K. D. and Seshaiah, P. (2000). Salivary gland development in Drosophila melanogaster. Mech. Dev. 92,5 -17.[CrossRef][Medline]
Armknecht, S., Boutros, M., Kiger, A. A., Nybakken, K., Mathey-Prevot, B. and Perrimon, N. (2005). High-throughput RNA interference screens in Drosophila tissue culture cells. Methods Enzymol. 392,55 -73.[CrossRef][Medline]
Balavoine, G. and Adoutte, A. (1998). One or
three Cambrian radiations? Science
280,397
-398.
Beachy, P. A., Varkey, J., Young, K. E., von Kessler, D. P., Sun, B. I. and Ekker, S. C. (1993). Cooperative binding of an Ultrabithorax homeodomain protein to nearby and distant sites. Mol. Cell. Biol. 13,6941 -6856.[Abstract]
Bermingham, J. R., Martinez-Arias, A., Petitt, M. G. and Scott,
M. P. (1990). Different patterns of transcription from the
two Antennapedia promoters during Drosophila embryogenesis.
Development 109,553
-566.
Bienz, M., Saari, G., Tremml, G., Muller, J., Zust, B. and Lawrence, P. A. (1988). Differential regulation of Ultrabithorax in two germ layers of Drosophila. Cell 53,567 -576.[CrossRef][Medline]
Brand, A. H., Manoukian, A. S. and Perrimon, N. (1994). Ectopic expression in Drosophila. In Methods in Cell Biology (ed. L. S. B. Goldstein and E. Fyrberg), pp. 635-654. New York: Academic Press.
Busturia, A., Vernos, I., Macias, A., Casanova, J. and Morata, G. (1990). Different forms of Ultrabithorax proteins generated by alternative splicing are functionally equivalent. EMBO J. 9,3551 -3555.[Abstract]
Campos-Ortega, J. A. and Hartenstein, V. (1985). The Embryonic Development of Drosophila melanogaster. Berlin: Springer-Verlag.
Capovilla, M. and Botas, J. (1998). Functional
dominance among Hox genes: repression dominates activation in the regulation
of Dpp. Development 125,4949
-4957.
Capovilla, M., Brandt, M. and Botas, J. (1994). Direct regulation of decapentaplegic by Ultrabithorax and its role in Drosophila midgut morphogenesis. Cell 76,461 -475.[CrossRef][Medline]
Carroll, S. B., Layman, R. A., McCutcheon, M. A., Riley, P. D. and Scott, M. P. (1986). The localization and regulation of Antennapedia protein expression in Drosophila embryos. Cell 47,113 -122.[CrossRef][Medline]
Castelli-Gair, J., Greig, S., Micklem, G. and Akam, M.
(1994). Dissecting the temporal requirements for homeotic gene
function. Development
120,1983
-1995.
Catron, K. M., Zhang, H., Marshall, S. C., Inostroza, J. A., Wilson, J. M. and Abate, C. (1995). Transcriptional repression by Msx-1 does not require homeodomain DNA-binding sites. Mol. Cell. Biol. 15,861 -871.[Abstract]
Chan, H. M. and La Thangue, N. B. (2001).
p300/CBP proteins: HATs for transcriptional bridges and scaffolds.
J. Cell Sci. 114,2363
-2373.
Chan, S. K., Jaffe, L., Capovilla, M., Botas, J. and Mann, R. S. (1994). The DNA binding specificity of Ultrabithorax is modulated by cooperative interactions with extradenticle, another homeoprotein. Cell 78,603 -615.[CrossRef][Medline]
Chan, S. K., Popperl, H., Krumlauf, R. and Mann, R. S. (1996). An extradenticle-induced conformational change in a HOX protein overcomes an inhibitory function of the conserved hexapeptide motif. EMBO J. 15,2476 -2487.[Abstract]
Chang, C. P., Shen, W. F., Rozenfeld, S., Lawrence, H. J., Largman, C. and Cleary, M. L. (1995). Pbx proteins display hexapeptide-dependent cooperative DNA binding with a subset of Hox proteins. Genes Dev. 9,663 -674.[Abstract]
Chariot, A., Princen, F., Gielen, J., Merville, M. P., Franzoso,
G., Brown, K., Siebenlist, U. and Bours, V. (1999a).
IkappaB-alpha enhances transactivation by the HOXB7 homeodomain-containing
protein. J. Biol. Chem.
274,5318
-5325.
Chariot, A., van Lint, C., Chapelier, M., Gielen, J., Merville, M. P. and Bours, V. (1999b). CBP and histone deacetylase inhibition enhance the transactivation potential of the HOXB7 homeodomain-containing protein. Oncogene 18,4007 -4014.[CrossRef][Medline]
Cohen, B., Wimmer, E. A. and Cohen, S. M. (1991). Early development of leg and wing primordia in the Drosophila embryo. Mech. Dev. 33,229 -240.[CrossRef][Medline]
Cohen, S. M. (1990). Specification of limb development in the Drosophila embryo by positional cues from segmentation genes. Nature 343,173 -177.[CrossRef][Medline]
Cohen, S. M., Brönner, G., Küttner, F., Jürgens, G. and Jäckle, H. (1989). Distal-less encodes a homoeodomain protein required for limb development in Drosophila.Nature 338,432 -434.[CrossRef][Medline]
Fasano, L., Roder, R., Core, N., Alexandre, E., Vola, C., Jacq, B. and Kerridge, S. (1991). The gene teashirt is required for the development of Drosophila embryonic trunk segments and encodes a protein with widely spaced zinc finger motifs. Cell 64,63 -79.[CrossRef][Medline]
Featherstone, M. (2003). Hox proteins and their co-factors in transcriptional regulation. In Murine Homeobox Gene Control of Embryonic Patterning and Organogenesis, Vol.13 (ed. Lufkin, T.), pp. 1-42. Amsterdam: Elsevier.[CrossRef]
Florence, B. and McGinnis, W. (1998). A genetic
screen of the Drosophila X chromosome for mutations that modify
Deformed function. Genetics
150,1497
-1511.
Galant, R. and Carroll, S. B. (2002). Evolution of a transcriptional repression domain in an insect Hox protein. Nature 415,910 -913.[CrossRef][Medline]
Galant, R., Walsh, C. M. and Carroll, S. B.
(2002). Hox repression of a target gene:
extradenticle-independent, additive action through multiple monomer binding
sites. Development 129,3115
-3126.
Gebelein, B., Culi, J., Ryoo, H. D., Zhang, W. and Mann, R. S. (2002). Specificity of Distalless repression and limb primordia development by abdominal Hox proteins. Dev. Cell 3,487 -498.[CrossRef][Medline]
Gebelein, B., McKay, D. J. and Mann, R. S. (2004). Direct integration of Hox and segmentation gene inputs during Drosophila development. Nature 431,653 -659.[CrossRef][Medline]
Glass, C. K., Rose, D. W. and Rosenfeld, M. G. (1997). Nuclear receptor coactivators. Curr. Opin. Cell Biol. 9,222 -232.[CrossRef][Medline]
Gonzalez-Reyes, A. and Morata, G. (1990). The developmental effect of overexpressing a Ubx product in Drosophila embryos is dependent on its interactions with other homeotic products. Cell 61,515 -522.[CrossRef][Medline]
Grenier, J. K. and Carroll, S. B. (2000).
Functional evolution of the Ultrabithorax protein. Proc. Natl.
Acad. Sci. USA 97,704
-709.
Hafen, E., Levine, M. and Gehring, W. J. (1984). Regulation of Antennapedia transcript distribution by the bithorax complex in Drosophila.Nature 307,287 -289.[CrossRef][Medline]
Hittinger, C. T., Stern, D. L. and Carroll, S. B.
(2005). Pleiotropic functions of a conserved insect-specific HOX
peptide motif. Development
132,5261
-5270.
Hughes, C. L. and Kaufman, T. C. (2002). Hox genes and the evolution of the arthropod body plan. Evol. Dev. 4,459 -499.[CrossRef][Medline]
Immerglück, K., Lawrence, P. A. and Bienz, M. (1990). Induction across germ layers in Drosophila mediated by a genetic cascade. Cell 62,261 -268.[CrossRef][Medline]
Irvine, K. D., Botas, J., Jha, S., Mann, R. S. and Hogness, D.
S. (1993). Negative autoregulation by Ultrabithorax
controls the level and pattern of its expression.
Development 117,387
-399.
Johnson, A. D., Poteete, A. R., Lauer, G., Sauer, R. T., Ackers, G. K. and Ptashne, M. (1981). lambda Repressor and cro components of an efficient molecular switch. Nature 294,217 -223.[CrossRef][Medline]
Johnson, F. B., Parker, E. and Krasnow, M. A.
(1995). Extradenticle protein is a selective cofactor for the
Drosophila homeotics: Role of the homeodomain and YPWM amino acid
motif in the interaction. Proc. Natl. Acad. Sci. USA
92,739
-743.
Knoepfler, P. and Kamps, M. (1995). The pentapeptide motif of Hox proteins is required for cooperative DNA binding with Pbx1, physically contacts Pbx1, and enhances binding by Pbx1. Mol. Cell. Biol. 15,5811 -5819.[Abstract]
Kosman, D., Mizutani, C. M., Lemons, D., Cox, W. G., McGinnis,
W. and Bier, E. (2004). Multiplex detection of RNA expression
in Drosophila embryos. Science
305, 846.
Lewis, E. B. (1978). A gene complex controlling segmentation in Drosophila. Nature 276,565 -570.[Medline]
Li, X., Murre, C. and McGinnis, W. (1999).
Activity regulation of a Hox protein and a role for the homeodomain in
inhibiting transcriptional activation. EMBO J.
18,198
-211.
Manak, J. R., Mathies, L. D. and Scott, M. P. (1995). Regulation of a decapentaplegic midgut enhancer by homeotic proteins. Development 120,3605 -3619.
Mann, R. S. and Hogness, D. S. (1990). Functional dissection of Ultrabithorax protein in D. melanogaster.Cell 60,597 -610.[CrossRef][Medline]
Martinez, P., Lee, J. C. and Davidson, E. H. (1997). Complete sequence of SpHox8 and its linkage in the single Hox gene cluster of Strongylocentrotus purpuratus. J. Mol. Evol. 44,371 -377.[Medline]
Martinez-Arias, A. (1986). The Antennapedia gene is required and expressed in parasegments 4 and 5 of the Drosophila embryo. EMBO J. 5, 135-141.
McCormick, A., Core, N., Kerridge, S. and Scottt, M.
(1995). Homeotic response elements are tightly linked to
tissue-specific elements in a transcriptional enhancer of the
teashirt gene. Development
121,2799
-2812.
McGinnis, N., Ragnhildstveit, E., Veraksa, A. and McGinnis,
W. (1998). A cap `n' collar protein isoform contains a
selective Hox repressor function. Development
125,4553
-4564.
McGinnis, W. and Krumlauf, R. (1992). Homeobox genes and axial patterning. Cell 68,283 -302.[CrossRef][Medline]
Medina-Martínez, O. and Ramírez-Solis, R. (2003). In vivo mutagenesis of the Hoxb8 hexapeptide domain leads to dominant homeotic transformations that mimic the loss-of-function mutations in genes of the Hoxb cluster. Dev. Biol. 264, 77-90.[CrossRef][Medline]
Merabet, S., Kambris, Z., Capovilla, M., Berenger, H., Pradel, J. and Graba, Y. (2003). The hexapeptide and linker regions of the Abd-A Hox protein regulate its activating and repressive functions. Dev. Cell 4,761 -768.[CrossRef][Medline]
Muller, J., Thuringer, F., Biggin, M., Zust, B. and Bienz, M. (1989). Coordinate action of a proximal homeoprotein binding site and a distal sequence confer the Ultrabithorax expression pattern in the visceral mesoderm. EMBO J. 8,4143 -4151.[Abstract]
Neuteboom, S., Peltenburg, L., van Dijk, M. and Murre, C.
(1995). The hexapeptide motif LFPWMR in Hoxb-8 is required for
cooperative DNA binding with Pbx1 and Pbx2 proteins. Proc. Natl.
Acad. Sci. USA 92,9166
-9170.
Panzer, S., Weigel, D. and Beckendorf, S. K.
(1992). Organogenesis in Drosophila melanogaster:
embryonic salivary gland determination is controlled by homeotic and
dorsoventral patterning genes. Development
114, 49-57.
Passner, J. M., Ryoo, H. D., Shen, L., Mann, R. S. and Aggarwal, A. K. (1999). Structure of a DNA-bound Ultrabithorax-Extradenticle homeodomain complex. Nature 397,714 -719.[CrossRef][Medline]
Pearson, J. C., Lemons, D. and McGinnis, W. (2005). Modulating Hox gene functions during animal body patterning. Nat. Rev. Genet. (in press).
Perutz, M. F. (1989). Mechanisms of cooperativity and allosteric regulation in proteins. Q. Rev. Biophys. 22,139 -237.[Medline]
Phelan, M. L., Rambaldi, I. and Featherstone, M. S. (1995). Cooperative interactions between HOX and PBX proteins mediated by a conserved peptide motif. Mol. Cell. Biol. 15,3989 -3997.[Abstract]
Piper, D. E., Batchelor, A. H., Chang, C. P., Cleary, M. L. and Wolberger, C. (1999). Structure of a HoxB1-Pbx1 heterodimer bound to DNA: role of the hexapeptide and a fourth homeodomain helix in complex formation. Cell 96,587 -597.[CrossRef][Medline]
Rauskolb, C. and Wieschaus, E. (1994). Coordinate regulation of downstream genes by extradenticle and the homeotic selector proteins. EMBO J. 13,3561 -3569.[Abstract]
Remacle, S., Abbas, L., De Backer, O., Pacico, N., Gavalas, A.,
Gofflot, F., Picard, J. J. and Rezsöhazy, R. (2004).
Loss of function but no gain of function caused by amino acid substitutions in
the hexapeptide of Hoxa1 in vivo. Mol. Cell. Biol.
24,8567
-8575.
Reuter, R., Panganiban, G. E. F., Hoffmann, F. M. and Scott, M.
P. (1990). Homeotic genes regulate the spatial expression of
putative growth factors in the visceral mesoderm of Drosophila
embryos. Development
110,1031
-1040.
Roder, V. and Kerridge, S. (1992). The role of
the teashirt gene in trunk segmented identity in Drosophila.Development 115,1017
-1033.
Ronshaugen, M., McGinnis, N. and McGinnis, W. (2002). Hox protein mutation and macroevolution of the insect body plan. Nature 415,914 -917.[CrossRef][Medline]
Ryoo, H. D. and Mann, R. S. (1999). The control of trunk Hox specificity and activity by Extradenticle. Genet. Dev. 13,1704 -1716.
Saffman, E. E. and Krasnow, M. A. (1994). A
differential response element for the homeotics at the Antennapedia
P1 promoter of Drosophila. Proc. Natl. Acad. Sci. USA
91,7420
-7424.
Saleh, M., Rambaldi, I., Yang, X.-J. and Featherstone, M. S.
(2000). Cell signaling switches HOX-PBX complexes from repressors
to activators of transcription mediated by histone deacetylases and histone
acetyltransferases. Mol. Cell. Biol.
20,8623
-8633.
Schughart, K., Utset, M. F., Awgulewitsch, A. and Ruddle, F.
H. (1988). Structure and expression of Hox-2.2, a
murine homeobox-containing gene. Proc. Natl. Acad. Sci.
USA 85,5582
-5585.
Shanmugam, K., Featherstone, M. S. and Saragovi, H. U.
(1997). Residues flanking the HOX YPWM motif contribute to
cooperative interactions with PBX. J. Biol. Chem.
272,19081
-19087.
Smolik-Utlaut, S. M. (1990). Dosage
requirements of Ultrabithorax and bithoraxoid in the
determination of segment identity in Drosophila melanogaster.Genetics 124,357
-366.
Subramaniam, V., Bomze, H. M. and Lopez, A. J.
(1994). Functional differences between Ultrabithorax protein
isoforms in Drosophila melanogaster: evidence from elimination,
substitution and ectopic expression of specific isoforms.
Genetics 136,979
-991.
Sun, B., Hursh, D. A., Jackson, D. and Beachy, P. A. (1995). Ultrabithorax protein is necessary but not sufficient for full activation of decapentaplegic expression in the visceral mesoderm. EMBO J. 14,520 -535.[Abstract]
Tan, X. X., Bondos, S., Li, L. and Matthews, K. S. (2002). Transcription activation by Ultrabithorax Ib protein requires a predicted alpha-helical region. Biochemistry 41,2774 -2785.[CrossRef][Medline]
Tremml, G. and Bienz, M. (1989). Homeotic gene expression in the visceral mesoderm of Drosophila embryos. EMBO J. 8,2677 -2685.[Abstract]
Vachon, G., Cohen, B., Pfeifle, C., McGuffin, M. E., Botas, J. and Cohen, S. M. (1992). Homeotic genes of the bithorax complex repress limb development in the abdomen of the Drosophila embryo through the target gene Distal-less. Cell 71,437 -450.[CrossRef][Medline]
White, R. A. H. and Wilcox, M. (1984). Protein products of the bithorax complex in Drosophila. Cell 39,163 -171.[CrossRef][Medline]
Yaron, Y., McAdara, J. K., Lynch, M., Hughes, E. and Gasson, J.
C. (2001). Identification of novel functional regions
important for the activity of HOXB7 in mammalian cells. J.
Immunol. 166,5058
-5067.
Zhang, H., Catron, K. M. and Abate-Shen, C.
(1996). A role for the Msx-1 homeodomain in tanscriptional
regulation: Residues in the N-terminal arm mediate TATA binding protein
interaction and transcriptional repression. Proc. Natl. Acad. Sci.
USA 93,1764
-1769.
Zhao, J. J., Lazzarini, R. A. and Pick, L. (1996). Functional dissection of the mouse Hox-a5 gene. EMBO J. 15,1313 -1322.[Abstract]
Related articles in Development: