1 Department of Biological Sciences, Graduate School of Science, University of
Tokyo
2 Core Research for Evolutional Science and Technology (CREST), Japan Science
and Technology Corporation (JST), 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033,
Japan
Author for correspondence (e-mail:
m_taira{at}biol.s.u-tokyo.ac.jp).
Accepted 21 May 2003
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SUMMARY |
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Key words: Xenopus, Spemann organizer, Protein-protein interactions, stoichiometry, E3 ubiquitin ligase, Proteasome, RING finger protein, LIM homeodomain protein, XRnf12/RLIM, Ldb1, Xlim-1
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INTRODUCTION |
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LIM-HD proteins have two highly conserved LIM domains in their N termini,
which are involved in protein-protein interactions
(Dawid et al., 1998;
Bach, 2000
). The LIM domains
have been shown to produce negative regulatory effect on Xlim-1
(Taira et al., 1994b
). Binding
of cofactors to the LIM domains is thought to relieve the inhibitory effect of
the LIM domains on Xlim-1; Ldb1 (also known as NLI and CLIM2) being one such
factor (Agulnick et al., 1996
;
Jurata et al., 1996
;
Bach et al., 1997
). Ldb1
contains a self-dimerization domain and a LIM interaction domain
(Jurata and Gill, 1997
;
Breen et al., 1998
), and a
dimer of Ldb1 has been shown to bridge two LIM-HD molecules (Lhx3/Lim3, Isl1,
or Isl3) to form a tetrameric complex
(Jurata et al., 1998
). In the
Drosophila wing disc, overexpression of Chip, the Drosophila
ortholog of Ldb1, results in wing malformation, which is rescued by
co-expression of the LIM-HD protein Apterous, suggesting that the
stoichiometric ratio between LIM-HD and Ldb proteins is critical for LIM-HD
activity (Fernandez-Funez et al.,
1998
; Milan and Cohen,
1999
; van Meyel et al.,
1999
; Rincon-Limas et al.,
2000
). Tetrameric complex formation has also been supported by the
observations that chimeric molecules in which the dimerization domain of Chip
or Ldb1 is fused to a LIM domain-deleted construct of Apterous or Xlim-1,
respectively, are as functional as co-expressed wild-type molecules
(Milan and Cohen, 1999
;
van Meyel et al., 1999
;
Rincon-Limas et al., 2000
;
Hiratani et al., 2001
). Thus,
LIM-HD proteins are likely to function as a tetrameric complex with Ldb1 in a
number of developmental contexts, but the mechanisms regulating the
stoichiometric ratio between LIM-HD factor and Ldb1 are largely unknown.
In mice, a novel regulator for the LIM-HD transcription factor Lhx3, RLIM
(also referred to as Rnf12 according to mouse gene nomenclature), has been
isolated and shown to be capable of suppressing the activity of Lhx3
(Bach et al., 1999). Rnf12
contains a RING finger motif at its C terminus, which is a conserved,
cysteine-rich, zinc-binding motif found in a diverse group of ubiquitin (Ub)
ligases that mediate the transfer of Ub to heterologous substrates
(Jackson et al., 2000
;
Joazeiro and Weissman, 2000
).
Ub ligases (E3) are determinants of target specificity in the protein
ubiquitination pathway. After Ub is transferred from a Ub-activating enzyme
(E1) to a Ub-conjugating enzyme (E2), Ub ligases (E3) promotes transfer of Ub
from Ub-conjugating enzyme (E2) to a specific target protein, which is
subsequently degraded by the proteasome
(Hershko and Ciechanover,
1998
; Glickman and
Ciechanover, 2002
). Suppression of LIM-HD factors by Rnf12
possibly relies on this activity, which has proved to be the case from recent
work by Ostendorff et al. (Ostendorff et
al., 2002
), in addition to its proposed role in recruiting the
histone deacetylase complex (Bach et al.,
1999
). Thus, Rnf12/RLIM appears to be a negative regulator for
LIM-HD/Ldb1 complexes.
To determine whether Rnf12 also plays any role in the regulation of Xlim-1, we isolated the Xenopus ortholog XRnf12 and examined its functional interactions with Xlim-1 and Ldb1 in early Xenopus development. Developmental expression analysis of XRnf12 in comparison with Xlim-1 and Ldb1 revealed that the three genes are co-expressed in the Spemann organizer, raising the possibility that XRnf12 does not simply function as a negative regulator for Xlim-1. Our biochemical and functional analyses show that XRnf12 initiates ubiquitinproteasome-dependent degradation of excess Ldb1 but not Ldb1 bound to Xlim-1 nor Xlim-1 itself, suggesting a role for XRnf12 in adjustment of Xlim-1/Ldb1 stoichiometry in the organizer by assuring proper Ldb1 expression levels.
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MATERIALS AND METHODS |
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Cloning of XRnf12
PCR with degenerate primers, dF4 and dR5, was carried out using a
Xenopus neurula (stage 17/18) cDNA library (J. Shinga and M.T.,
unpublished). Primers were designed based on the conserved amino acid
sequences in mouse and chick Rnf12: forward primer dF4,
5'-CA(A/G)AT(A/C/T)ATGACIGG(A/C/G/T)TT-(C/T)GG-3' (I, inosine),
which corresponds to the amino acid sequence QIMTGFG of XRnf12, and reverse
primer dR5, 5'-TT(A/G)TC(A/G/T)AT-(C/T)TG(C/T)TC(C/T)TT-3', which
corresponds to the sequence KEQIDN of XRnf12. An amplified 0.36 kb fragment
was cloned and used as a probe to screen a Xenopus gastrula cDNA
library (a kind gift from B. Blumberg).
Plasmid constructs
The following plasmids were constructed and used for mRNA injections. To
make pCS2+XRnf12, a PCR-amplified, NcoI-digested XRnf12 coding region
was subcloned into NcoI-StuI-digested pCS2+AdN
(Mochizuki et al., 2000).
pCS2+XRnf12(HC>AA) was generated by replacing His585 and Cys588 of XRnf12
with alanines using the Gene Editor in vitro Site-Directed Mutagenesis System
(Promega). pCS2+Xlim-1-FLAG contains the Xlim-1 coding region flanked by a
single FLAG epitope tag at the C terminus. pCS2+FLAGLdb1 contains the Ldb1
coding region downstream of a single FLAG epitope tag derived from pCS2+FTn
(formerly pCS2+FLAG) (Mochizuki et al.,
2000
). pCS2+FLAG-Ldb1
C was constructed by replacing Ldb1
with Ldb1
C [formerly Ldb1(1-291)]
(Hiratani et al., 2001
). For
pCS2+3HA-ubiquitin, a single copy of the Xenopus ubiquitin was
PCR-amplified from a Xenopus neurula cDNA library and subcloned into
EcoRI-XbaI-digested pCS2+3HA, which provides three
N-terminal HA epitope tags. Primer sequences were based on the second
ubiquitin repeat sequence of pXlgC20
(Dworkin-Rastl et al., 1984
)
and are as follows: forward,
5'-ggaattctATGCAGATCTTTGTAAAA-3' (lower case, linker
sequences; underline, restriction site); reverse,
5'-gctctagaCTAGCCACCCCTGAGCCGAAG-3'. pCS2+NLS-ABL60,
pCS2+NLS-CT239, and pCS2+NLS-CT261 contain amino acids 1-177, 239-403, and
261-403 of Xlim-1, respectively, downstream of the SV40 large T antigen NLS
(MAPKKKRKV). pCS2+HD34 contains amino acids 178-272 of Xlim-1. pCS2+LMO2
contains full-length mouse LMO2 amplified by PCR. pCS2+hRNF6, pCS2+hRNF13, and
pCS2+hRNF38 were constructed with pCS2+ by PCR amplification of the entire
coding region from cDNA clones (GenBank accession numbers: BC034688, BC009781,
and BC033786, respectively) provided by the Mammalian Gene Collection (MGC)
project (NIH) through Open Biosystems. pSP64-Xßm (Xenopus
ß-globin) has been described (Krieg
and Melton, 1984
).
The following plasmids were used for GST pull-down assays.
pGEX2TNEX-XRnf12C and pGEX2TNEX-XRnf12
N contain amino acids
1-283 and 282-616, respectively, of XRnf12 in pGEX2TNEX
(Hiratani et al., 2001
).
pGEX2T-Ldb1 contains full-length Ldb1 in pGEX2T. pGEX2T-ABL60,
pGEX2T-
C, pGEX2TCT239, pGEX2T-
NA, and pGEX2T-Xlim-1 contain
portions of Xlim-1 shown in Fig.
5E in pGEX2T. pGEX2T-HD27 has been described previously [formerly
GST/Xlim-1(HD27)] (Mochizuki et al.,
2000
). pSP64T-Xlim-5 was constructed as follows: the coding region
of Xlim-5 was obtained from pBluescript-KS(+)Xlim-5 plasmid
(Toyama et al., 1995
) by
PvuII-HincII digestion, and inserted into end-filled
BglII site of pSP64T (Krieg and
Melton, 1984
). pSP64T-Xlim-3 has been described previously
(Yamamoto et al., 2003
). All
constructs were verified by sequencing.
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Northern blot and RT-PCR analysis
Northern blot analysis was performed using the stored original blot from
the previous study (Hikasa and Taira,
2001). A 0.36 kb PCR fragment amplified with degenerate primers
dF4 and dR5, which contained the XRnf12C sequence, was cloned into
the pT7Blue vector (Novagen), excised with NdeI-EcoRI,
gel-purified, and used as a XRnf12 probe. RT-PCR analysis was done as
described previously (Osada et al.,
2003
).
Nuclear ß-gal staining, whole-mount in situ hybridization and
sectioning
For lineage tracing, nß-gal mRNA transcribed from linearized
pCS2+nß-gal was co-injected and visualized by Red-Gal (Research Organics)
staining. Whole-mount in situ hybridization was carried out as described
previously (Harland, 1991;
Shinga et al., 2001
). For
hemisections, rehydrated embryos were embedded in 2% low melt agarose in
1x PBS containing 0.3 M sucrose and 0.05% Triton X-100, and sectioned
with a razor blade before hybridization as described previously
(Lee et al., 2001
).
Digoxigenin-labeled antisense RNA probes were transcribed from Xlim-1
(Taira et al., 1992
),
gsc (Cho et al.,
1991
), chd (Sasai et
al., 1995
), Xotx2
(Pannese et al., 1995
),
XPAPC (Kim et al.,
1998
), cer
(Bouwmeester et al., 1996
),
XFKH1 (Dirksen and Jamrich,
1992
), Xnot (von
Dassow et al., 1993
), Mix.1
(Rosa, 1989
) and Xbra
(Smith et al., 1991
) plasmid
templates. PCR-amplified template containing a T7 promoter sequence was used
for dkk1 (Shibata et al.,
2001
). pBluescript-SK(-)XRnf12 and pBluescript-SK(-)XLdb1
(Agulnick et al., 1996
) were
used for XRnf12 and Ldb1 probes, respectively.
Albino embryos were stained slightly more intensively than usual for better
interior staining and embedded in paraffin wax and sectioned. Nuclei were
stained with DAPI (4',6-diamidino-2-phenylindole) as described
previously (Shibata et al.,
2001).
Protein extraction
Embryos were collected at the gastrula stage (stage 10.5 or 11) and
homogenized in 10 µl of homogenizing buffer (50 mM Tris-HCl, pH 8.0, 50 mM
KCl, 20% glycerol, 1 mM EDTA, 1 mM EGTA, 5 mM DTT, 1 mM PMSF) per embryo (or
2-2.5 µl per animal cap), and the supernatant was collected after
centrifugation. For the dispersed cell experiments, mRNA-injected embryos were
cultured in Ca2+/Mg2+-free 1x MBS containing 0.2%
BSA and 50 µg/ml gentamicin sulfate in agarose-coated dishes, and vitelline
membranes were removed at stage 6. Then, 50 µM (final) MG-132 (Peptide
Institute), or an equal volume of DMSO for the negative control, was added to
the medium and embryos were dispersed into single cells by gentle agitation.
Cells were collected at the gastrula stage for preparation of cell lysates.
Protein concentrations of the lysates were determined by a Protein Assay kit
(Bio-Rad).
Western blot analysis and whole-mount immunostaining
For western blotting, equivalent amounts of total proteins were separated
by SDS-PAGE, transferred to a PVDF membrane, and visualized either by chemical
luminescence using the ECL system (Amersham Pharmacia) or by alkaline
phosphatase staining using NBT/BCIP. Whole-mount immunostaining was done
essentially as described previously
(Hiratani et al., 2001),
except for the use of ImmunoPure Metal-Enhanced DAB Substrate Kit (Pierce) for
staining. Bisection of gastrula embryos were done as described previously
(Lee et al., 2001
). Antibodies
used are as follows: anti-FLAG (M2, Sigma), anti-HA (12CA5, Roche),
anti-ß-tubulin (clone tub 2.1, Sigma), anti-Ldb1/CLIM2 (N-18, Santa
Cruz), anti-Ldb1/NLI (Jurata et al.,
1996
), horseradish peroxidase (HRP)-conjugated anti-mouse Ig
antibody from sheep (Amersham Pharmacia), alkaline phosphatase-conjugated
anti-mouse IgG antibody from goat (Promega), HRP-conjugated anti-goat IgG from
donkey (Santa Cruz), and HRP-conjugated anti-rabbit Ig from donkey (Amersham
Pharmacia).
Coimmunoprecipitation assay
Coimmunoprecipitation assays were done essentially as described previously
(Watanabe and Whitman, 1999),
with slight modifications. Cell lysates were collected as described above
except for the use of lysis buffer [20 mM Tris-HCl (pH 8.0), 1 mM EDTA, 10%
glycerol, 8 mM DTT, 40 µg/ml leupeptin, 20 µg/ml aprotinin, 1 mM PMSF]
containing 0.1% NP-40. Equivalent amounts of lysates (100-200 µl) were
incubated with 1 µg of anti-FLAG antibody for 1 hour at 4°C, then added
with 40 µl of protein G-agarose (Roche), and incubated for another 30
minutes at 4°C. After being washed 5 times with lysis buffer sequentially
containing each of the following: (1) 0.1% NP-40, (2) 0.4 M NaCl, (3) 0.5%
NP-40, (4) 0.2 M NaCl and 0.25% NP-40, and (5) nothing, SDS sample buffer was
added and the bound proteins were eluted from beads by boiling. The eluted
proteins were separated by SDS-PAGE, followed by western analysis.
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RESULTS |
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Because the N-terminal region of Rnf12 binds to both LIMHD proteins and
Ldb1/CLIM2 (Bach et al., 1999),
we performed GST pull-down experiments with XRnf12, Xenopus LIM-HD
proteins, and Ldb1. As expected, the N-terminal region [amino acids (aa)
1-283] of XRnf12 interacted with Xlim-1, Xlim-3, Xlim-5, and Ldb1
(Fig. 1C, GSTXRnf12
C),
while the C-terminal region (aa 282-616) of XRnf12 showed little or no
interaction with any of them (Fig.
1C, GST-XRnf12
N). We also found that the N-terminal region
(aa 1-291) of Ldb1 is sufficient for the interaction with XRnf12
C
(Fig. 1C, Ldb1
C). Thus,
XRnf12 possesses characteristics similar to mouse Rnf12 in terms of
protein-binding specificity.
Expression of XRnf12 overlaps with that of Xlim-1
and Ldb1 in the gastrula mesoderm
To evaluate temporal expression patterns of the XRnf12 genes, we
carried out northern analysis using a 359 base probe of XRnf12C,
which shares 93% and 99% identity at the nucleotide level with XRnf12
and XRnf12B, respectively, assuming that any of the three
XRnf12 genes could be detected with this probe. Two maternal
XRnf12 transcripts of different sizes (3.0 and 3.5 kb) were detected
at the cleavage stage (Fig.
2A). While the 3.0 kb transcript disappeared at the gastrula stage
(Fig. 2A, stage 11), the 3.5 kb
transcript is maintained at relatively constant levels throughout early
embryogenesis, with a slight increase at the late gastrula stage
(Fig. 2A, stage 12.5). Judging
from their size differences, the 3.0 kb and 3.5 kb transcripts may represent
XRnf12 and XRnf12B/C genes, respectively.
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The similarity of XRnf12 and Ldb1 expression at the
gastrula stage led us to further compare their expression during
embryogenesis. After the gastrula stage, the expression of both transcripts is
gradually restricted to tissues originated from the ectoderm, the
neuroectoderm, neural crest and epidermis (data not shown), and subsequently
to the neural tube as well as the head and the tailbud region
(Fig. 2E-J). Additional
expression of Ldb1 is seen in the pronephric field, and the
profundal-trigeminal placodal area (pPrV)
(Schlosser and Northcutt,
2000) at the neural groove to neural tube stages
(Fig. 2H,I, arrowhead).
Although not as localized, XRnf12 also appears to be present in the
pPrV area (Fig. 2E,F,
arrowhead). At the tailbud stage, expression of both genes is detected in the
pronephric region and branchial arches
(Fig. 2G,J). Cross sections of
tailbud embryos confirmed their colocalization in the epidermis, neural crest,
neural tube and head mesenchyme (Fig.
2K-P). These results show that the expression domains of
XRnf12 and Ldb1 largely overlap throughout early
embryogenesis, suggesting that the two genes may function in a common
regulatory process rather than having distinct roles independent of each other
(see Discussion).
XRnf12 suppresses secondary axis formation elicited by Xlim-1/Ldb1
and antagonizes organizer activity upon overexpression
Because Xlim-1, Ldb1 and XRnf12 transcripts colocalize in
the gastrula mesoderm, we next asked whether XRnf12 could affect the
axis-inducing activity of Xlim-1/Ldb1
(Agulnick et al., 1996). As
shown in Fig. 3A, co-expression
of XRnf12 markedly suppressed secondary axis formation elicited by
Xlim-1/Ldb1, while ß-globin, as negative control, had no effect. Notably,
point mutations in the RING finger, which is supposed to abolish its zinc
binding activity (Saurin et al.,
1996
), almost totally abolished the inhibitory action of XRnf12
(Fig. 3A, XRnf12(HC>AA)). We
further examined the effect of overexpression of XRnf12 in the dorsal marginal
zone. While XRnf12(HC>AA) had only a small effect (9% headless or cyclopic,
n=118; Fig. 3B), overexpression of XRnf12 resulted in reduced head structures (52% headless or
cyclopic, n=171; Fig.
3C), suggesting that XRnf12 is likely to interfere with organizer
function through RING finger-dependent activity. This was also confirmed by
downregulation of organizer gene expression by XRnf12 as described below.
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We further examined whether XRnf12 mediates Ldb1 degradation by the
proteasome. We co-expressed FLAG-Ldb1, Xlim-1, and XRnf12 ventrally, dispersed
embryos into single cells, and cultured them in the presence of the proteasome
inhibitor MG-132 until the gastrula stage. XRnf12-mediated reduction of
FLAG-Ldb1 showed suppression in the presence of MG-132
(Fig. 4E, lane 6). MG-132
treatment alone had little or no effect on FLAG-Ldb1 expression
(Fig. 4E, lanes 3,4). These
results suggest that XRnf12 causes degradation of Ldb1 by the proteasome, also
confirming the recent report on its mouse counterpart
(Ostendorff et al., 2002).
To test the specificity of XRnf12, we analyzed three other related RING finger proteins, human RNF6, RNF38 and RNF13, for their ability to reduce Ldb1 expression levels. These three human proteins were chosen because they showed highest similarity to XRnf12 from database searching. Amino acid sequence identities of hRNF6, hRNF38 and hRNF13 to XRnf12 are 42%, 22% and 16% in entire proteins, and 80%, 51%, and 55% in the RING fingers, respectively. As shown in Fig. 4F, hRNF6 caused reduction in FLAG-Ldb1 expression levels, which seemed reasonable because of the relatively high sequence similarity between hRNF6 and XRnf12. That hRNF38 also caused reduction in FLAG-Ldb1 expression levels, albeit to a lesser extent, was somewhat unexpected, but the observed interaction between Ldb1 and hRNF38 in a GST pull-down assay provides a possible explanation for the activity of hRNF38 as well as hRNF6 (Fig. 4G). Human RNF13, which is the least similar to XRnf12 in the entire region of the three Rnf12-related proteins tested, did not interact with Ldb1 or affect its expression (Fig. 4F,G). These results suggest that not all Rnf12-related RING finger proteins can be involved in Ldb1 degradation, indicating that there is some degree of specificity in the activity of XRnf12.
Xlim-1 suppresses XRnf12-mediated degradation of Ldb1 through
interaction with Ldb1
The fact that XRnf12 mediates ubiquitin-proteasome-dependent degradation of
Ldb1 raises important questions. First, how is the activity of Xlim-1/Ldb1,
which is apparently required in the organizer, assured in the presence of
XRnf12? Second, what is the functional significance of Ldb1 degradation by
XRnf12?
A clue to the first question came from our observation that co-expression of Xlim-1 reproducibly caused an increase in the expression level of FLAG-Ldb1 (Fig. 4B, compare lanes 2 and 4). As shown in Fig. 5A, we further confirmed that Xlim-1 dose-dependently increased the expression level of FLAG-Ldb1 (lanes 8-12), which may result from interfering with endogenous XRnf12. Moreover, we found that Xlim-1 suppressed XRnf12-mediated reduction of FLAG-Ldb1 levels in a dose-dependent manner (Fig. 5A, lanes 3-7). While the expression level of FLAG-Ldb1 became saturated in our range of Xlim-1 dosages in the absence of XRnf12 (Fig. 5A, lanes 11,12), it continued to increase at the same Xlim-1 dosages in the presence of XRnf12 (Fig. 5A, lanes 6,7). These results suggest that Xlim-1 suppresses the degradation of Ldb1 caused by exogenous XRnf12 and possibly endogenous XRnf12, implying mutual interactions between the three proteins.
To define the region in Xlim-1 required for this suppression, we expressed
a series of Xlim-1 deletion constructs together with FLAG-Ldb1 and XRnf12, and
examined the expression level of FLAG-Ldb1. Of all the constructs tested, only
the LIM domain-containing ABL60 efficiently suppressed degradation of Ldb1
caused by XRnf12 (Fig. 5B, lane
5). Mouse LMO2 (Foroni et al.,
1992) also suppressed degradation of Ldb1 caused by XRnf12
(Fig. 5C), although the
interpretation of this result is complicated a little by the reported
ubiquitin ligase activity of Rnf12 toward LMO2 (see Discussion). We conclude
from our results that LIM domains are sufficient for the inhibition of XRnf12
activity.
We next defined the XRnf12-binding region in Xlim-1 by GST pull-down assay
using a series of GST-Xlim-1 constructs and 35S-labeled XRnf12.
Contrary to the reported LIM domain-binding of mouse Rnf12
(Bach et al., 1999), the LIM
domain-containing ABL60 showed virtually no interaction with XRnf12
(Fig. 5D). In contrast, the
homeodomain-containing HD27 and
NA showed weak interactions with XRnf12
(Fig. 5D). Thus, the
homeodomain-containing region (aa 178-265) of Xlim-1 is necessary and
sufficient for the interaction with XRnf12
(Fig. 5E). Under our
experimental conditions, GST-Xlim-1 showed a weaker affinity for XRnf12 than
GST-Ldb1 (Fig. 5D). We also
noticed that XRnf12 interacts with itself through the C-terminal region (aa
282-616) (Fig. 5D,
GST-XRnf12
N), suggesting that XRnf12 may form a homodimeric
complex.
Taken together, the LIM domain-containing region of Xlim-1, which is
required for the interaction with Ldb1
(Agulnick et al., 1996;
Breen et al., 1998
) but not
XRnf12, is sufficient for the suppression of XRnf12-mediated Ldb1 degradation.
This suggests that binding of Xlim-1 to Ldb1 is a requisite for the
suppression. We further tested this possibility by using FLAG-Ldb1
C,
which does not contain the LIM interaction domain
(Jurata and Gill, 1997
;
Breen et al., 1998
).
FLAG-Ldb1
C contains the region required for ubiquitination
(Fig. 4D) and RING
finger-dependent degradation by XRnf12
(Fig. 5F, lanes 2,3,6). In
striking contrast to FLAG-Ldb1, XRnf12-mediated degradation of
FLAG-Ldb1
C was not suppressed by Xlim-1 or other Xlim-1 constructs
tested in Fig. 5B
(Fig. 5F lanes 4,5 and data not
shown), further supporting our hypothesis that binding of Xlim-1 to Ldb1 is
required for the suppression. These results suggest that Ldb1 escapes
degradation by XRnf12 upon association with Xlim-1, providing a plausible
explanation of the way in which Xlim-1/Ldb1 could exert its effect in the
presence of XRnf12 in the organizer.
No apparent dorsal-to-ventral (D/V) difference in the expression
levels of the Ldb1 protein
Because Xlim-1 is enriched in the dorsal mesoderm, our results suggest that
Ldb1 may be subject to selective degradation by XRnf12 in the ventrolateral
mesoderm, thus contributing to further spatial restriction of Xlim-1 activity
to the dorsalmost region. We addressed this question by using an anti-Ldb1
antibody, N-18, which recognizes a peptide sequence mapping at the conserved N
terminus of vertebrate Ldb1. Specificity of the antibody to the
Xenopus Ldb1 protein was assessed by immunoblotting of
Xenopus embryo extracts, which expressed a single band of a predicted
size of approximately 46 kDa. Furthermore, exogenous Ldb1 expression by mRNA
injection enhanced this 46 kDa band, whereas addition of blocking peptides at
1 µg/ml eliminated immunoreactivity, reflecting specificity (not shown). We
also confirmed that endogenous Ldb1 expression in animal caps is downregulated
by overexpression of XRnf12 in a RING-dependent fashion
(Fig. 6A), consistent with the
results using exogenous Ldb1 constructs (Figs
4,
5).
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Excess Ldb1 suppresses the expression of Xlim-1 target genes, which
is rescued by co-expression of XRnf12
We next hypothesized that the functional significance of Ldb1 degradation
by XRnf12 may be to eliminate excess Ldb1 molecules. We first examined the
effect of Ldb1 overexpression on the axis-inducing activity of Xlim-1
(Fig. 7). As previously
reported (Agulnick et al.,
1996; Hiratani et al.,
2001
), co-expression of Xlim-1 (0.25 ng mRNA/embryo) and Ldb1 (0.5
ng mRNA/embryo) initiated secondary axis formation. Notably, excess amounts of
Ldb1 (1.0 to 4.0 ng/embryo) inhibited this activity dose-dependently, whereas
increasing the dose of Xlim-1 effectively suppressed the inhibitory action of
excess amounts of Ldb1 (4.0 ng mRNA/embryo)
(Fig. 7). These results suggest
that the stoichiometry of Xlim-1 and Ldb1 is important for the exertion of
their function, and that excess Ldb1 molecules are deleterious to Xlim-1. It
is also possible that excess Ldb1 interferes with LIM domain-dependent
association of Xlim-1 with other proteins, if any.
|
|
Ldb1 overexpression affects the maintenance phase of organizer gene
expression
Our previous study suggests a role for Xlim-1 in the maintenance phase of
organizer gene expression rather than initiation
(Mochizuki et al., 2000).
Therefore, we analyzed the effect of Ldb1 overexpression on gsc
expression during late blastula to mid gastrula stages (stages 9.5, 10, 10.5,
and 11) to see if Ldb1 overexpression primarily affected the maintenance
phase. Embryos were injected dorsally with Ldb1 mRNA and RNA was then
isolated from these embryos at stages 9.5, 10, 10.5 and 11 and subjected to
RT-PCR analysis. As shown in Fig.
9, downregulation of gsc by Ldb1 overexpression was
prominent at stages 10.5 and 11, whereas the effect was not as prominent at
stages 9.5 and 10. Consistent with the results in
Fig. 8, the reduction of
gsc expression at stages 10.5 and 11 was suppressed by co-expression
of XRnf12. These data support the idea that Ldb1 overexpression primarily
affected the maintenance phase of gsc expression elicited by Xlim-1
rather than initiation.
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DISCUSSION |
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We have shown that overexpression of Ldb1 or XRnf12 affects expression of candidate Xlim-1 target genes, gsc, chd, Xotx2, XPAPC and cer (Fig. 8, group 1). However, we have also noticed that overexpression of either XRnf12 (group 2) or Ldb1 (group 3) affects the expression of genes that have not been shown to be regulated by Xlim-1 and Ldb1 (Fig. 8, groups 2 and 3). Possible explanations for this observation with the group 2 and 3 genes are as follows: (1) The group 2 genes may be regulated by a Ldb1-containing complex which is disrupted by XRnf12 but not by excess amounts of Ldb1, and (2) Mix.1 and dkk1 in group 3 may be regulated by a Ldb1-independent complex, but this complex is disrupted by excess Ldb1 through Ldb1-interacting components in the complex, similar to the case of the cer gene. Curiously, inhibition of dkk1 expression by Ldb1 was also rescued by XRnf12(HC>AA) (Fig. 8H). Binding of XRnf12(HC>AA) to Ldb1 may suffice to suppress the effect of excess Ldb1 in this case. Although these possibilities remain to be elucidated, it should be emphasized that the expression of candidate Xlim-1 target genes, but not other genes examined, are affected by both Ldb1 and XRnf12. In addition, because the effect of excess Ldb1 on the expression of group 3 genes was relatively small compared to that on group 1 genes, it seems that Ldb1 overexpression primarily affects dorsal mesodermal gene expression by Xlim-1. Most importantly, all the genes examined show normal expression upon co-expression of Ldb1 and XRnf12, supporting the requirement of proper expression levels of Ldb1, which may be assured in the presence of XRnf12.
Roles of XRnf12 in other developmental contexts
XRnf12 and Ldb1 are expressed in a similar fashion
throughout early developmental stages (Fig.
2). This is reminiscent of the term `synexpression group'
(Niehrs and Pollet, 1999),
supporting the close functional interactions between Ldb1 and XRnf12. Because
Ldb1 seems to escape degradation by binding to Xlim-1, we first assumed a
dorsal-to-ventral gradient of Ldb1 protein expression, according to the
distribution of Xlim-1 protein (Karavanov
et al., 1996
). This seems not to be the case, as we did not
observe dorsal-toventral difference in Ldb1 expression levels
(Fig. 6B,C). This result raises
the possibility that Ldb1-interacting proteins other than Xlim-1, which could
bind and protect Ldb1 from degradation, are present in the lateral and ventral
regions as well as in the animal pole region
(Fig. 10). This possibility
could be explained by the presence of XLMO4 in the ventrolateral mesoderm of
the Xenopus embryo (J. L. Gomez-Skarmeta, personal communication) and
Xlim-5 in the animal pole region (Toyama
et al., 1995
). In the ventrolateral mesoderm, Ldb1 may participate
in a transcriptional regulatory complex that contains XLMO4 and perhaps GATA
proteins, by analogy with the case of Ldb1, LMO2, and GATA-1 in blood
development (Osada et al.,
1995
; Wadman et al.,
1997
). Because mouse LMO2, which is related to XLMO4, can block
degradation of Ldb1 by XRnf12 (Fig.
5C), it is possible that LMO proteins [LMO1-4 (reviewed by
Bach, 2000
)] also utilize
XRnf12 to acquire proper LMO/Ldb1 stoichiometry.
During the course of neuronal differentiation in the neural tube, several
LIM-HD proteins are expressed to generate a so-called LIM code that is thought
to define neuronal identity (Lumsden,
1995; Tanabe and Jessell,
1996
). LIM-HD proteins are also involved in brain development
(Sheng et al., 1996
;
Porter et al., 1997
;
Zhao et al., 1999
).
Interestingly, it has recently been shown that LIM-HD/Ldb1 stoichiometry
appears to be important for the specification of motor neuron and interneuron
identity by Lhx3/Islet-1/Ldb1 and Lhx3/Ldb1, respectively
(Thaler et al., 2002
). Because
XRnf12 and Ldb1 are co-expressed in the brain and the spinal cord
(Fig. 2), XRnf12 may contribute
to LIM-HD/Ldb1 stoichiometry in these regions as well.
Regulation of protein stability by mutual interactions
In the Drosophila wing disc, complex formation between Apterous
and Chip/dLDB attenuates proteasome-dependent degradation of Apterous and
stabilizes Apterous protein (Weihe et al.,
2001). Interestingly, we also noticed an increase in the
expression level of Xlim-1 by Ldb1 co-expression
(Fig. 4A, compare lanes 2,3
with lanes 4,5), suggesting that a similar mechanism for stabilization of
Xlim-1 protein exists in vertebrates. Therefore, Xlim-1 may be more
susceptible to proteasome-mediated degradation by some unknown factor(s) when
not bound to Ldb1. This may contribute to the establishment of proper
Xlim-1/Ldb1 stoichiometry dorsally, or may contribute to complete elimination
of Xlim-1 protein ventrally (Fig.
10). Notably, similar protein stabilization by heterodimerization
has been reported for the yeast transcription factors MAT
2/MATa1
(Johnson et al., 1998
) and for
the Drosophila homeodomain proteins Homothorax and Extradenticle
(Abu-Shaar and Mann, 1998
).
Cofactor exchange or maintenance of stoichiometry?
Ostendorff et al. proposed a model showing that Rnf12 mediates degradation
of Ldb1/CLIM2 in complex with Lhx3 on the promoter region, thereby replacing
Ldb1/CLIM2 with other cofactors
(Ostendorff et al., 2002).
However, this does not accord with our results that showed suppression of
XRnf12-mediated degradation of Ldb1 by excess amounts of Xlim-1. It was also
reported that Rnf12 regulates the activity of Lhx3 by recruiting the histone
deacetylase complex (Bach et al.,
1999
), and that this regulation is independent of its RING finger
(Ostendorff et al., 2002
). In
contrast to Lhx3, the inhibitory effect of XRnf12 overexpression on the
activity of Xlim-1 solely relied on its RING finger, suggesting that ubiquitin
ligase activity toward Ldb1 is the primary cause of the inhibition. Thus,
there seems to be some difference in the way in which Rnf12 regulates
different LIM-HD transcription factors. Alternatively, the difference may be
the result of the different experimental systems: Xenopus embryos and
cell cultures. We also did not observe interaction between XRnf12 and the LIM
domains of Xlim-1, contrary to the reported binding of mouse Rnf12 to LIM
domains of LIM-HD proteins, Lhx2, Lhx3 and Isl-1
(Bach et al., 1999
). This may
imply a difference in the binding affinity between Rnf12 and different LIM-HD
proteins. Alternatively, XRnf12 might actually interact with the LIM domains
of Xlim-1, although this could not be observed under our experimental
conditions. Thus, the molecular basis underlying the difference in
Rnf12-mediated regulation of Lhx3 and Xlim-1 remains to be elucidated. The
functional significance of the reported ubiquitination and degradation of LMO
proteins by Rnf12/RLIM (Ostendorff et al.,
2002
), and the mechanisms by which Rnf12/RLIM distinguishes LMO
proteins from LIM-HD proteins, are also important questions to be
answered.
Nevertheless, the results so far seem to be in good agreement about the close functional interactions between LIM-HD, Ldb1 and Rnf12 proteins. We believe that our results expand the knowledge of LIM-HD regulation and provide an attractive possibility that multimeric transcriptional regulatory complex such as LIM-HD/Ldb1 complexes are regulated in a similar way in which selective degradation of excess transcription factors or adapter proteins occurs through the ubiquitinproteasome pathway for the establishment of their proper stoichiometry.
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ACKNOWLEDGMENTS |
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Footnotes |
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