1 Department of Molecular Biology and Functional Genomics, DIBIT-H San Raffaele,
Via Olgettina 58, 20132 Milano, Italy
2 Molecular Medicine Unit, Institute of Child Health, 30 Guilford Street, London
WC1N 1EH, UK
3 Clinical Genetics Unit, Birmingham Women's Hospital, Edgbaston, Birmingham B15
2TG, UK
4 Department of Animal Biology, University of Modena and Reggio Emilia, Via
Campi 213/d, Modena 41100, Italy
Author for correspondence (e-mail:
zappavigna.vincenzo{at}hsr.it)
Accepted 13 January 2003
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SUMMARY |
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Key words: Hox genes, Limb malformations, Missense mutation, DNA binding, Posterior prevalence
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INTRODUCTION |
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To date, only two Hox genes have been proven to be mutated in human
malformation syndromes, HOXD13 in synpolydactyly (SPD) and
HOXA13 in hand-foot-genital syndrome (reviewed by
Goodman, 2002). SPD is a rare
dominantly inherited limb malformation, which is characterised by syndactyly
between the third and fourth fingers and between the fourth and fifth toes,
with a partial or complete extra digit in the syndactylous web. Typical SPD is
caused by expansions of a 15-residue polyalanine tract in the N-terminal
region of HOXD13 (Muragaki et al.,
1996
; Akarsu et al.,
1996
; Goodman et al.,
1997
). The mutant protein is thought to act as a dominant
negative, interfering functionally with wild-type HOXD13 and other 5'
HOXD proteins expressed in the autopod
(Zakany and Duboule, 1996
;
Bruneau et al., 2001
). An
atypical form of SPD, which is characterised by a distinctive foot phenotype,
has also been identified in four unrelated families. Three families harbour
different frameshifting deletions in HOXD13
(Goodman et al., 1998
;
Calabrese et al., 2000
), which
are predicted to result in truncated proteins unable to bind DNA, while the
fourth family harbours a missense mutation in helix II of the HOXD13
homeodomain (R31W), which is predicted to destabilise the homeodomain
(Debeer et al., 2002
). All
four mutations are thus likely to cause functional haploinsufficiency for
HOXD13. The typical and atypical forms of SPD produced by these two classes of
mutations are very similar, however, leaving open the possibility that other
HOXD13 mutations may produce unexpected phenotypes.
Although the identification of human limb malformations caused by HOXD13 mutations provide a unique opportunity to gain insight into the role of HOXD13 in limb development, no previous studies have characterised the effects of these mutations at a molecular level. We describe a six-generation family in which a unique combination of brachydactyly and central polydactyly co-segregates with a missense mutation in helix III of the HOXD13 homeodomain (I47L). We have compared the functions of the HOXD13(I47L) mutant protein with those of wild-type HOXD13 and a HOXD13 mutant unable to bind DNA. We show that the I47L substitution does not produce a dominant-negative effect or a gain of function, but instead impairs DNA binding at a subset of sites recognised by wild-type HOXD13, causing a selective loss of function. Consistently, retrovirus-mediated overexpression of wild-type HOXD13 in the chick autopod upregulates chick EphA7, a putative downstream target of Hoxa13, but overexpression of HOXD13(I47L) does not. Interestingly, the two mutant proteins produce more severe phenotypes than the wild-type protein in proximal regions of the chick limb, suggesting that functional suppression of anterior Hox proteins by more posterior ones does not require DNA-binding activity and may be mediated solely by protein:protein interactions.
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MATERIALS AND METHODS |
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Cell culture and transfections
P19 mouse embryonic carcinoma cells were cultured in Minimum Essential
Medium Alpha (Invitrogen) supplemented with 10% foetal calf serum (Celbio), 2
mM L-glutamine (Invitrogen), 100 U/ml penicillin and 100 µg/ml
streptomycin. Primary chick embryo fibroblasts (CEF cells) were cultured in
Dulbecco's Medium (Life Technologies). Transfections were carried out by
CaPO4 precipitation (Di Nocera
and Dawid, 1983). In a typical experiment, 10 µg of pTHCR
reporter plasmid, 0.5-2.5 µg of expression construct and 0.25 µg of
pCMV-ßgal (Clontech) as internal control were used per 10 cm dish.
Forty-eight hours after transfection, cells were washed, lysed and assayed for
luciferase and ß-galactosidase expression
(Zappavigna et al., 1994
).
Immunoblots of extracts from transfected cells showed that the expression
constructs produced identical amounts of HOXD13 and HOXD13(I47L) (data not
shown).
Electrophoretic mobility shift assays (EMSAs)
Full-length HA-tagged HOXD13 and HOXD13(I47L) proteins were synthesised in
vitro using the TNT-coupled transcription/translation system (Promega),
diluted in 13 µl of -buffer (20% glycerol, 20 mM KCl, 2 mM
MgCl2, 0.2 mM EDTA, 0.5 mM DTT) and pre-incubated with 100 ng
poly-(dI-dC) in a total volume of 20 µl 1x binding buffer (0.1 M KCl,
2 mM MgCl2, 4 mM spermidine, 0.1 mg/ml BSA) for 15 minutes on ice.
The amount of reticulocyte lysate used was adjusted to normalise for
translated protein content. 20,000-50,000 cpm of 32P-labelled probe
were added and samples were incubated for 30 minutes on ice. Reactions were
separated on 6% polyacrylamide gels in 0.5x TBE, which were dried and
exposed to Kodak X-OMAR film at 80°C. GST-HOXD13HD,
GST-HOXD13HD(I47L) and GST-HOXD13HD(IQN) fusion proteins were expressed in
E. coli, purified according to established methods and analysed by
SDS-PAGE and Coomassie staining. Probe sequences are given in Figs
5 and
6. The SelD13 probe was
generated by annealing the SelD13 oligonucleotide pool
(Fig. 5C) with a 10-fold molar
excess of the primer Sel3 (5'-GGCGAGATCTCTCGAGGG-3') and extending
with Klenow polymerase in the presence of
-[32P]-dCTP.
Quantitative evaluation of the DNA-binding efficiencies was performed using an
Amersham Bioscience Densitometer.
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Avian retrovirus production, microinjection, cartilage staining and
whole-mount in situ hybridisation
CEF cells were transfected by CaPO4 precipitation with 20 µg
of the RCAS-HOXD13, RCAS-HOXD13(I47L) or RCAS-HOXD13(IQN) retroviral
constructs to generate virus stocks, which were harvested, concentrated and
titrated on CEF cells (Morgan et al.,
1992). A titre of approximately 1x108 cfu/ml was
obtained for each virus. Fertilised eggs were incubated at 37°C for 1.5
days until stage 10 (Hamburger and
Hamilton, 1992
), when virus was introduced into the prospective
right limb area by a series of five to ten closely spaced injections
(Morgan et al., 1992
). The
eggs were returned to the incubator and harvested at stages 28-34. For
cartilage staining, embryos were fixed in 96% ethanol for 3-5 days, stained in
Alcian Blue (15 mg Alcian Blue 8GX (Sigma) in 80 ml 96% ethanol and 20 ml
glacial acetic acid) for 24 hours, and rinsed twice in 96% ethanol for 2 days,
before being cleared first in 1% KOH on ice and then in 2% KOH:glycerol
(20:80). Injected and control limbs were dissected and photographed through a
dissecting microscope. Cartilage lengths were measured using an ocular
reticule. Whole-mount in situ hybridisation
(Wilkinson and Nieto, 1993
)
was performed using a digoxigenin-labelled antisense mRNA probe to chick
EphA7 (Araujo and Nieto,
1997
).
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RESULTS |
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The I47L mutation impairs HOXD13's ability to activate transcription
at the HCR element
No naturally occurring HOXD13 DNA-binding sites have been characterised, as
none of the target genes of HOXD13 have yet been identified. To examine
whether the I47L substitution affects the ability of HOXD13 to control
transcription, we used the HCR sequence
(Fig. 4C), a highly conserved
92 bp regulatory element derived from the HOXD9 promoter, which can
mediate transcriptional activation by HOXD9 and HOXD10
(Zappavigna et al., 1991). To
compare HOXD13(I47L) with a HOXD13 mutant that is completely unable to bind
DNA, we also generated an artificial mutant, HOXD13(IQN), carrying alanine
substitutions at positions 47(I), 50(Q) and 51(N) of the homeodomain. These
three highly conserved residues in the recognition helix make crucial
base-specific DNA contacts (Gehring et
al., 1994
), so we predicted that replacing them with alanine would
abolish DNA binding altogether. We transiently co-transfected P19 cells with a
luciferase reporter construct driven by the HCR sequence (pTHCR), together
with increasing amounts of SV40-driven constructs expressing HOXD13,
HOXD13(I47L) or HOXD13(IQN). Although HOXD13 increased basal reporter activity
five- to sixfold, HOXD13(I47L) increased it only about 1.5-fold, and
HOXD13(IQN) had virtually no effect (Fig.
4A). Thus, the I47L substitution severely compromises the ability
of HOXD13 to activate transcription through the HCR element, suggesting that
it significantly impairs the capacity of the protein to bind DNA.
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The I47L mutation impairs the ability of HOXD13 to bind DNA
To investigate whether the weak transcriptional activation mediated by
HOXD13(I47L) at the HCR reflects defective DNA binding, we performed EMSAs
using oligonucleotide probes derived from this element. The HCR contains
several sites recognised in vitro by 5' HOXD proteins and supports the
formation of multiple retarded complexes with different stoichiometries in
EMSAs with HOXD10 (Zappavigna et al.,
1991). We first used bases 1-55 of the HCR (HCR
;
Fig. 5A), which contain four
sets of TAAT and/or TTAT motifs that could potentially be bound by HOX
proteins. While HOXD13 bound HCR
efficiently, forming three different
retarded complexes (Fig. 5A,
lanes 3-5), HOXD13(I47L) bound more weakly, producing detectable complexes
only at higher protein concentrations (Fig.
5A, lanes 7 to 9). We next used bases 3-25 of the HCR (HCR I;
Fig. 5B), because they contain
an 8 bp sequence (5'-TTTTATTA-3') that is identical to the
consensus binding site previously reported for Abd-B and Hoxa10
(5'-TTTTAT(T/G)(A/G)-3')
(Ekker et al., 1994
;
Benson et al., 1995
) and
differs at just two positions (underlined) from the consensus binding site
previously reported for Hoxd13 (5'-TTTTACGA-3')
(Shen et al., 1997
). HOXD13
formed a strong retarded complex with HCR I
(Fig. 5B, lane 3), whereas
HOXD13(I47L) bound only weakly (Fig.
5B, lane 4). We subsequently used bacterially expressed, purified
GST fusions of the HOXD13 and HOXD13(I47L) homeodomains [GST-HOXD13HD and
GST-HOXD13HD(I47L)] to determine their dissociation constants (Kd)
at this site in EMSAs, obtaining Kd values of
5x108 M and 3x107 M
respectively (data not shown). Thus, HOXD13(I47L) fails to recognise at least
some sites bound by the wild-type protein. This suggests that the I47L
mutation produces a loss of function, but leaves open the possibility that it
produces a gain of function by changing binding site specificity.
To explore these alternatives, we designed an oligonucleotide probe (SelD13; Fig. 5C) with 12 central bases derived from HCR I (GTTTTATTAGGG), but with the ATTAG sequence replaced by five random bases (GTTTTNNNNNGG), thus generating a large pool of sequence variants. We expected that a straightforward switch in binding specificity would result in equally efficient binding by HOXD13 and HOXD13 (I47L), as the different optimal binding sequences would be equally represented in the pool. As shown in Fig. 5C, when the SelD13 probe was incubated with increasing amounts of the purified GST-homeodomain fusion proteins, GST-HOXD13HD bound efficiently at all concentrations tested (lanes 2-6) whereas GST-HOXD13HD(I47L) bound only at higher concentrations (lanes 7-11). GST-HOXD13HD(IQN) bound marginally even at the highest concentration tested (lanes 12-16), indicating, as predicted, that HOXD13HD(IQN) does not recognise any site in the pool efficiently. The intermediate binding levels seen with HOXD13HD(I47L) are not consistent with a straightforward switch in binding specificity, but could have occurred either because the mutant protein binds more weakly than the wild-type protein at all sites in the pool, or because the mutant binds a smaller subset of sites in the pool.
HOXD13(I47L) binds a subset of the sites recognised by HOXD13
To distinguish between these possibilities, we performed binding site
selection assays with purified GST-HOXD13HD and GST-HOXD13HD(I47L) using the
SelD13 oligonucleotide pool. After five rounds of selection, the sequences of
100 oligonucleotides selected by each protein were examined. Of 100 sites
selected by wild-type HOXD13 (Fig.
6A), 48 had a TTAT core, followed in 36 by TGG. Only 2 had
a TAAT core. 32 had a TTAC core, followed in 25 by GAG. The
remaining 18 had a TAAC core, followed in 17 by GAG. Wild-type HOXD13
therefore appears to have an equal preference for two distinct sites, one
(site 1) with T in the fourth core position (TTTTATTGG) and
the other (site 2) with C in the fourth core position
(TTT(T/A)ACGAG) (Fig.
6B). By contrast, HOXD13(I47L) recognised a more restricted set of
sites. Of 100 sites selected (Fig.
6A), only three had a TTAT or TAAT core. 73 had a
TTAC core, followed in 47 by GAG, while 20 had a TAAC core,
followed in 11 by GAG. HOXD13(I47L) therefore appears to recognise one of the
two sites selected by wild-type HOXD13 (site 2), while almost completely
failing to recognise the other (site 1)
(Fig. 6B).
To confirm these findings, we performed EMSAs using probes containing site 1 (with the TTAT core) and the two variants of site 2 (with TTAC and TAAC cores). As shown in Fig. 6C, HOXD13 bound all three probes with comparable efficiency (lanes 2-4, 9-11 and 16-18). HOXD13(I47L) bound the TTAT-containing probe much more weakly than the wild-type protein (16- and ninefold less at 5 and 10 ng respectively, lanes 5-6), but bound the TTAC- and TAAC-containing probes with the same efficiency (0.9-fold less TTAC at 5 and 10 ng, lanes 12-13; 1.3- and 1.1-fold less TAAC at 5 and 10 ng respectively, lanes 19-20). Thus, the I47L mutation does not result in recognition of a novel DNA-binding sequence. Instead, it causes a selective impairment of DNA-binding ability, producing a marked reduction in affinity at one class of sites recognised by the wild-type protein, but no loss of affinity at the other.
Misexpression of HOXD13(I47L) in developing chick limbs does not
affect the digits but produces more severe proximal abnormalities than
HOXD13
To explore the effects of the I47L mutation further in vivo, we used the
developing chick limb, a well-established model system for studying limb
development in vertebrates. The availability of replication-competent
retroviral vectors that permit gene transfer into avian cells has made it
possible to manipulate chick limb development genetically in ovo
(Morgan and Fekete, 1996). We
therefore generated recombinant retroviral vectors expressing full-length
HOXD13, HOXD13(I47L) or HOXD13(IQN), and injected concentrated retroviral
suspensions of comparable titres into the prospective right hindlimb field of
stage 10 chick embryos in ovo. Control experiments using a retroviral
construct expressing alkaline phosphatase
(Fekete and Cepko, 1993
)
confirmed that the entire right leg bud was infected at high frequency (data
not shown). Embryos were harvested at stage 32-33 (day 7.5-8) and stained with
Alcian Blue to allow visualisation of cartilaginous skeletal elements. The
left uninjected limbs were used as internal controls. The resulting phenotypes
are summarised in Table 1 and
illustrated in Fig. 7.
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Embryos misexpressing HOXD13(I47L) (Fig.
7B,C) likewise had no phalangeal abnormalities (except one embryo
out of 38, which had an extra anterior digit). The cartilages of the stylopod,
zeugopod and proximal autopod, however, were more severely shortened,
especially the tibia, which on average was reduced to 54% of its normal length
(Table 1). In 16% of embryos,
the morphology of the tibia was also altered from a typical long bone
cartilage to a rounded cartilage, with bowing of the fibula, sometimes
producing a complete inversion of normal limb posture
(Fig. 7C). In addition, 16% of
embryos had extra cartilages in the zeugopod
(Fig. 7B,C). Neither these
tibial changes nor ectopic cartilages were ever observed in embryos
misexpressing HOXD13 (Goff and Tabin,
1997) (this work).
Embryos misexpressing HOXD13(IQN) (Fig. 7D,E) again had no phalangeal abnormalities, but the proximal cartilages, especially the femur and tibia, were more severely shortened than in embryos misexpressing HOXD13, and the metatarsals were shorter than in embryos misexpressing either HOXD13 or HOXD13(I47L) (Table 1). However, no instances of altered tibial morphology or ectopic cartilages were observed in over 100 embryos examined. Strikingly, the phenotype produced by a HOXD13 mutant incapable of binding DNA is thus qualitatively similar to but quantitatively more severe than that produced by HOXD13.
Overexpression of HOXD13 upregulates EphA7, while
overexpression of HOXD13(I47L) does not
In Hoxa13/ mice, EphA7
expression is significantly reduced in the condensing mesenchyme of the
digits, carpals and tarsals, but not completely absent, suggesting that this
ephrin receptor is a downstream target not only of Hoxa13 but perhaps also of
other Hox proteins expressed in the developing autopod, like Hoxd13
(Stadler et al., 2001). To
investigate whether HOXD13 controls EphA7 expression, and, if so,
whether the I47L mutation affects this activity, we analysed chick
EphA7 expression in chick limbs overexpressing HOXD13, HOXD13(I47L)
or HOXD13(IQN). In control stage 28 hindlimbs, EphA7 was expressed in
the perichondrium of the phalangeal mesenchymal condensations
(Fig. 8A,C,E), as reported
previously in E13.5 mice (Stadler et al.,
2001
). Overexpression of HOXD13 markedly increased EphA7
expression in the perichondrium but produced no significant ectopic expression
(Fig. 8B). Overexpression of
HOXD13(I47L) or HOXD13(IQN), however, had no effect upon EphA7 levels
(Fig. 8D,F). Thus, while
HOXD13, like Hoxa13, upregulates EphA7 expression in the autopod,
neither HOXD13(I47L) nor HOXD13(IQN) retains this activity.
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DISCUSSION |
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The substitution in question alters the isoleucine residue at position 47
of the HOXD13 homeodomain to leucine. In the Antennapedia, engrailed and HOXB1
homeodomain/DNA complexes, I47 methyl groups make van der Waals contacts both
with the C5 methyl group of the thymine at position 4 of the Hox core binding
site (TAAT) and with C8 of the adenine at position 3 (TAAT)
(Gehring et al., 1994;
Fraenkel and Pabo, 1998
;
Piper et al., 1999
). In
keeping with this crucial role in DNA binding, residue 47 is highly conserved
(Banerjee-Basu et al., 2001
).
Seven out of the eight Drosophila Hox proteins and all but two of the
39 vertebrate Hox proteins have isoleucine at this position, the exceptions
all being group 2 paralogs (Drosophila proboscipedia, vertebrate
Hoxa2 and Hoxb2), which have valine, another non-polar branching amino acid
(Banerjee-Basu et al., 2001
).
Most other homeodomain proteins also have isoleucine or valine, although
different amino acids occur in some atypical homeodomains
(Banerjee-Basu and Baxevanis,
2001
; Banerjee-Basu et al.,
2001
). The only two reported instances of leucine at position 47
are in highly divergent proteins, PaHB2 of the Norway spruce (Picea
abies) and the bE1-bE7 mating type proteins of the smut fungus
(Ustiligo maydis) (GenBank Accession Numbers AAL83725, AAA63553-6 and
CAA38000-2).
No naturally occurring mutations affecting homeodomain residue 47 have
previously been reported (D'Elia et al.,
2001). An artificial I47A substitution in engrailed reduces DNA
binding affinity 10- to 20-fold in vitro
(Ades and Sauer, 1995
), while
an I47Q substitution in the Para-Hox protein IPF1 abolishes DNA binding in
vitro and greatly reduces transcriptional activation in transfected cells
(Lu et al., 1996
). A V47L
substitution in the POU homeodomain protein Oct2 also abolishes DNA binding in
vitro (Stepchenko et al.,
1997
). No I47L substitution, however, has yet been analysed.
HOXD13(I47L) exhibits a selective loss of DNA-binding ability
Previous studies have shown that the most 5' Hox protein in
Drosophila, Abd-B, selects sites with a TTAT core
(Ekker et al., 1994), whereas
the 5' vertebrate Hox proteins select sites with a TTAT or TTAC core
(Benson et al., 1995
;
Shen et al., 1997
). We found
that HOXD13 displayed an equal preference for two distinct consensus sites,
one containing a TTAT core (TTTTATTGG), and the other
containing a TTAC or TAAC core (TTT(T/A)ACGAG). This second
site is identical to the consensus Hoxd13 binding site obtained by Shen et al.
(TTTTACGAG) (Shen et
al., 1997
), except at position 4, where we observed a 64:36 T:A
ratio whereas Shen et al. observed a strong preference for T. HOXD13(I47L)
selected only the second of these sites (TTT(T/A)ACGAG),
perhaps reflecting the inability of L47 to make the contacts normally made by
I47 with a T residue in the fourth core position. Thus, the mutant protein,
rather than recognising a novel binding site, appears to have lost the ability
to recognise one class of site bound by the wild-type protein while retaining
the ability to recognise the other.
Consistent with these findings, HOXD13(I47L) bound the TTTTACGAG
and TTTAACGAG sequences with the same affinity as wild-type HOXD13
in EMSAs, but displayed a significantly lower affinity for the
TTTTATTGG sequence. The impaired capacity of HOXD13(I47L) to
recognise TTAT- and TAAT-containing sites was further confirmed using the HCR
element, which contains multiple TTAT and TAAT motifs bound by several
5' HOXD proteins, but only one TTAC motif
(Zappavigna et al., 1991).
Although HOXD13 bound strongly and specifically to several of these sites in
EMSAs, HOXD13(I47L) bound only weakly, with an affinity for one of them (HCR
I, TTTTATTAG) about sixfold lower than that of the wild-type
protein. Moreover, while HOXD13 activated transcription through the
full-length HCR, HOXD13(I47L) did not. These observations suggest that
HOXD13(I47L) is unable to regulate some of the genes regulated by wild-type
HOXD13.
HOXD13 upregulates EphA7 expression but HOXD13(I47L) does not
Signalling between Eph receptors and their ephrin ligands plays a key role
in regulating many developmental processes and is probably a downstream
effector of several 3' Hoxa and Hoxb genes [reviewed in
(Frisen et al., 1999)].
Moreover, Hoxa13 is necessary for normal EphA7 expression
levels in the autopod (Stadler et al.,
2001
). We found that misexpression of HOXD13 in chick limbs
increased chick EphA7 expression in the perichondrium of the digital
condensations, showing that HOXD13, like Hoxa13, can upregulate
EphA7. Interestingly, ectopic expression of chick EphA7 was
not induced, suggesting that additional factors are required for
EphA7 transcription. Misexpression of HOXD13(I47L), however, had no
effect on chick EphA7 levels, showing that the I47L mutation indeed
impairs the capacity of HOXD13 to regulate one of its downstream targets.
Although no other HOXD13 targets have yet been identified, our finding that
HOXD13(I47L) retains the ability in vitro to bind some sites bound by HOXD13
suggests that it can still regulate at least some of these targets. It may
thus partially or completely fail to regulate a subset of the genes normally
controlled by HOXD13, such as EphA7, while correctly regulating the
remainder, thus eliciting an unbalanced transcriptional response.
Misexpression of HOXD13(I47L) in chick limbs produces a phenotype
both quantitatively and qualitatively different to that produced by
HOXD13
To analyse the consequences of the I47L substitution in vivo, we used
retrovirus-mediated expression in the developing chick limb. When
overexpressed in the phalanges, where Hoxd13 is normally expressed,
HOXD13(I47L), like wild-type HOXD13 (Goff
and Tabin, 1997) (this work) and HOXD13(IQN), caused no defects.
Thus, HOXD13(I47L) does not interfere with the functions of endogenous Hoxd13
in vivo, or with distal autopod development, indicating that it does not act
by a dominant-negative or other gain-of-function mechanism. Consistently,
HOXD13(I47L) did not interfere with transcriptional activation by HOXD13 at
the HCR element in transiently transfected cells, and showed no gain or switch
in DNA-binding activity in vitro.
In proximal limb regions, however, whereas HOXD13 caused only mild
shortening of the long bone cartilages, HOXD13(I47L) produced severe
shortening, as well as striking abnormalities of zeugopod morphology,
including a change in the shape of the tibia from long to rounded cartilage
and the formation of ectopic cartilages. Misexpression of HOXD13(IQN) likewise
caused severe shortening of the proximal cartilages, but never produced the
abnormal zeugopod morphology observed with HOXD13(I47L). The phenotype caused
by HOXD13(I47L) in proximal limb regions is thus qualitatively as well as
quantitatively different from that produced by wild-type HOXD13, but is also
qualitatively different from that produced by HOXD13(IQN), which is completely
unable to bind DNA. This further strengthens the hypothesis that the I47L
substitution results in a selective rather than a generalised loss of
function. The additional zeugopod abnormalities caused by HOXD13(I47L)
probably reflect its ability to control only a subset of the genes normally
controlled by HOXD13, leading to an imbalance in the regulation of downstream
targets. Interestingly, misexpression of wild-type Hoxa13 in chick limbs
produces a similar zeugopod phenotype
(Yokouchi et al., 1995),
suggesting that some of the same targets may be involved.
A selective loss-of-function mechanism also accounts well for the novel brachydactyly-polydactyly syndrome produced by the mutation. Thus, the occasional central polydactyly in the hands, like that in typical and atypical SPD, probably reflects loss of transcriptional activity, while the impaired growth or absence of specific phalangeal and metaphalangeal bones, unaffected in both forms of SPD, probably reflects unbalanced regulation of the normal targets of HOXD13.
The ability of HOXD13 to interfere with proximal limb development
does not require DNA binding
The shortening of proximal limb skeletal elements produced by ectopic
expression of Hoxd13 in the chick (Goff
and Tabin, 1997) (this work) has also been observed in the mouse.
Thus, Hoxd13 expression in the prospective forearm causes shortening and
bowing of the radius and ulna both in mice with a Hoxd11/lacZ
transgene upstream of Hoxd13 (van
der Hoeven et al., 1996
;
Zakany and Duboule, 1999
) and
in Ulnaless mice (Herault et al.,
1997
; Peichel et al.,
1997
). Ectopic expression of Hoxa13 in the chick
(Yokouchi et al., 1995
) and of
Hoxd12 in the mouse (Knezevic et al.,
1997
) also produce proximal limb shortening. Similarly, ectopic
Hoxd gene expression resulting from mutations that perturb the regulation of
the Hoxd cluster may underlie some types of mesomelic dysplasia in humans
(Fujimoto et al., 1998
;
Spitz et al., 2002
;
Sugawara et al., 2002
).
In all these cases, the zeugopod shortening closely resembles that in
Hoxa11//Hoxd11+/
and
Hoxa11+//Hoxd11/
mice (Davis et al., 1995), and
appears to reflect functional interference with the endogenous group 11 Hox
proteins by the ectopic group 12 and/or 13 Hox proteins
(Zakany and Duboule, 1999
).
This is consistent with the well-established phenomenon known as phenotypic
suppression in Drosophila and posterior prevalence in vertebrates,
whereby the more posterior Hox proteins suppress the functions (but not the
expression) of more anterior Hox proteins when co-expressed in same region
(Bachiller et al., 1994
;
Duboule and Morata, 1994
). The
molecular basis for this functional hierarchy remains unclear, but competition
for shared sets of target genes and for DNA-binding partners and/or
transcriptional co-factors have been proposed
(Duboule and Morata,
1994
).
Strikingly, we found that the HOXD13(I47L) and HOXD13(IQN) mutants, the DNA-binding abilities of which are selectively and generally (respectively) impaired, caused more severe shortening of proximal limb cartilages than wild-type HOXD13. The capacity of HOXD13 to interfere with the growth-promoting functions of more anterior Hox proteins thus increases when its DNA-binding ability is impaired, suggesting that this interference is mediated by protein:protein interactions and is mitigated rather than exacerbated by the ability to regulate shared target genes. Our results therefore support a model in which posterior prevalence is based on competition for interacting partners and/or transcriptional co-factors, rather than on competition for targets.
Which region(s) of HOXD13 mediate these interactions? In the chick limb,
misexpression of a Hoxd13 protein lacking the first 98 amino acids produced
slightly less zeugopod shortening than full-length Hoxd13
(Goff and Tabin, 1997),
suggesting a possible role for the poorly characterised N-terminal region.
However, the zeugopod was severely shortened in mice homozygous for a
`knock-in' mutation in which the homeodomain of Hoxa11 was replaced with that
of Hoxa13 (Zhao and Potter,
2001
). Taken together with our findings, this result suggests that
the homeodomain itself may be sufficient to mediate posterior prevalence by
interacting with DNA-binding partners and/or co-factors without binding DNA.
Further work will be required to characterise the protein motifs and factors
involved.
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ACKNOWLEDGMENTS |
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Footnotes |
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