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Molecular and Cellular Biology, February 2001, p. 1360-1369, Vol. 21, No. 4
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.4.1360-1369.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
The Polycomb Group Protein EED Interacts with YY1, and Both
Proteins Induce Neural Tissue in Xenopus
Embryos
David P. E.
Satijn,
Karien M.
Hamer,
Jan
den
Blaauwen, and
Arie P.
Otte*
Swammerdam Institute for Life Sciences, BioCentrum
Amsterdam, University of Amsterdam, 1018 TV Amsterdam, The
Netherlands
Received 17 August 2000/Returned for modification 14 September
2000/Accepted 27 October 2000
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ABSTRACT |
Polycomb group (PcG) proteins form multimeric protein complexes
which are involved in the heritable stable repression of genes. Previously, we identified two distinct human PcG protein complexes. The
EED-EZH protein complex contains the EED and EZH2 PcG proteins, and the
HPC-HPH PcG complex contains the HPC, HPH, BMI1, and RING1 PcG
proteins. Here we show that YY1, a homolog of the
Drosophila PcG protein pleiohomeotic (Pho), interacts
specificially with the human PcG protein EED but not with proteins of
the HPC-HPH PcG complex. Since YY1 and Pho are DNA-binding proteins,
the interaction between YY1 and EED provides a direct link between the
chromatin-associated EED-EZH PcG complex and the DNA of target genes.
To study the functional significance of the interaction, we expressed
the Xenopus homologs of EED and YY1 in
Xenopus embryos. Both Xeed and XYY1 induce an ectopic
neural axis but do not induce mesodermal tissues. In contrast, members
of the HPC-HPH PcG complex do not induce neural tissue. The exclusive,
direct neuralizing activity of both the Xeed and XYY1 proteins
underlines the significance of the interaction between the two
proteins. Our data also indicate a role for chromatin-associated
proteins, such as PcG proteins, in Xenopus neural induction.
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INTRODUCTION |
During embryogenesis, the
fertilized egg develops into a complex organism with many
differentiated cell types. Maintenance of the differentiation status of
these cells requires a cellular memory system that is responsible
for the stable inheritance of gene expression (10). The
Polycomb group (PcG) and trithorax group (trxG) genes are part of such
a memory system, and in Drosophila they have been identified
as repressors (PcG) and activators (trxG), respectively (20,
33). Mutations in the PcG and trxG genes result in pleiotropic
defects, of which homeotic transformations are the most apparent. The
phenotypic defects in most of the PcG or trxG mutants become
apparent only relatively late during Drosophila development,
implying that these proteins indeed have a role in maintenance of cell
differentiation. In vertebrates, similar roles for the PcG and trxG
proteins in the maintenance of homeotic gene expression patterns, and
consequently changes in the body plan, have been observed in PcG
mutants (4, 29). However, mutations in some vertebrate PcG
genes result in very early defects in development. This is exemplified
by mutations in the eed (embryonic ectoderm development)
gene, the vertebrate homolog of the Drosophila PcG gene
extra sex combs (esc). The
eed
/ mouse shows very early defects in
development, resulting in gastrulation defects and lack of a node and
of neural tissue (28). This indicates the
involvement of PcG proteins in processes that precede maintenance of differentiation choices; it points towards involvement in
embryonic inductions of tissues.
PcG proteins have been found to interact with each other to form
multimeric, chromatin-associated protein complexes. Both in
Drosophila and in vertebrates, various components of PcG
complexes have been identified (27). Evidence has
accumulated that there are at least two distinct PcG complexes. The
human PcG homologs HPC2 (human Polycomb 2) (25), HPH
(human Polyhomeotic) (5), BMI1 (42), and
RING1 (24, 26) proteins belong to the HPC-HPH complex. The
human PcG homologs EED and EZH2 (Enhancer of Zeste 2) belong to the
second, EED-EZH PcG complex (30). The latter complex is
associated with histone deacetylase (HDAC) activity, through a specific
interaction between the EED and HDAC proteins (41).
The study of how PcG complexes regulate and interact with their target
genes at the level of DNA has been hampered by the fact that most PcG
proteins do not directly bind to DNA. The mouse homolog of the
Drosophila PcG protein posterior sex combs, mel-18, has been
shown to have DNA-binding activity (9), but not much is
known about interaction partners of mel-18. Recently the
Drosophila pleiohomeotic (Pho) protein has been found to
share extensive homology with the vertebrate transcription factor YY1,
a DNA-binding protein (1). This PcG protein could direct
either the HPC-HPH or the EED-EZH PcG complex to the DNA of target
genes. Here we show a specific interaction between YY1 and the EED PcG
protein, providing a direct link between the DNA of target genes and
the EED-EZH PcG protein complex.
To investigate the functional significance of this interaction we
studied the role of the Xenopus homologs of these proteins, Xeed and XYY1, in Xenopus embryogenesis. We found that both
Xeed and XYY1 directly induced neural tissue but were unable to induce mesodermal tissues. Our results indicate that the interaction between
EED and YY1 is significant for early developmental decisions. They also
suggest a novel role for chromatin-associated factors, such as the PcG
proteins, in Xenopus neural induction.
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MATERIALS AND METHODS |
Two-hybrid analysis.
Two-hybrid analysis was performed as
described previously (26). We cloned cDNAs encoding the
EZH2, HPH1, HPC2, EED, RING1, and Bmi1 proteins in frame with the GAL4
DNA-binding domain in the pAS2 vector (Clontech). The p53 protein
(pVA3) and simian virus 40 large antigen (pTD1) served as a positive
interaction control. The full-length YY1 cDNA was cloned in frame with
the GAL4 transactivation domain into the pGAD10 vector (Clontech). We
cotransformed plasmids into the yeast Y190 strain (Clontech) and plated
the transformants on medium lacking leucine, tryptophan, and histidine.
After 4 days the cells were grown to an optical density at 600 nm of
1.0 to 1.2. 
-Galactosidase activity was measured in
permeabilized cells as described previously (5).
GST pull-down assay.
We cloned the full-length
EED, EZH2, HPC2, and RING1
cDNAs into pGEX-2TK, thus creating glutathione S-transferase
(GST)-EED, GST-EZH2, GST-HPC2, and GST-RING1. We immobilized these GST
fusion proteins and GST protein alone on glutathione-Sepharose
4B. In vitro-translated,
[35S]methionine-labeled YY1 or Pho was
incubated with the GST fusion proteins in 250 µl of binding buffer
(phosphate-buffered saline with 1 mM EDTA; 1 mM dithiothreitol; 2 mM
phenylmethylsulfonyl fluoride [PMSF]; the protease inhibitors
leupeptin, benzamidine, and aprotinin; 1% [vol/vol] Triton X-100;
0.5% NP-40; and 1 mg of bovine serum albumin per ml) for 1 h at
4°C, under constant rotation. Finally, we analyzed the washed beads
on sodium dodecyl sulfate (SDS)-polyacrylamide gels, which were
subjected to autoradiography.
Immunoprecipitation assay.
We prepared nuclei from human
Ramos cells by 10 strokes with a glass Dounce homogenizer-pestle
in a buffer containing 20 mM HEPES (pH 7.0), 1.5 mM
MgCl2, 10 mM KCl, 0.5 mM dithiothreitol, and
0.5 mM PMSF. The nuclei were pelleted by centrifugation at 1,000 × g at 4°C for 10 min. Subsequently, the
nuclei were lysed in lysis buffer (250 mM NaCl, 0.1% NP-40, 50 mM
HEPES [pH 7.0], 5 mM EDTA, 0.5 mM dithiothreitol, 1 mM PMSF, and the
protease inhibitors leupeptin, benzamidine, and aprotinin). The lysate was sonicated two times with bursts of 15 s, and the supernatant was incubated with antibodies (indicated below) for 2 h at 4°C. Goat anti-rabbit immunoglobulin G antibody coupled to agarose beads
(Sigma) was added for 30 min at 4°C. The beads were washed six times,
and the immunoprecipitate (IP) was separated by SDS-polyacrylamide gel
electrophoresis and transferred to nitrocellulose. The blots were
probed with a rabbit antibody against EED, EZH2, or HPC2 or a mouse
monoclonal antibody against YY1 (SC-7872; Santa Cruz).
Plasmid constructions.
The 1,278-bp open reading frame of
Xeed was cloned into the pCS2+ vector (23). The XYY1 cDNA
was cloned by PCR, using primers based on the reported sequence
(21). Capped synthetic RNA was made by in vitro
transcription as described previously (18). To verify that
the synthesized mRNAs still retained the capacity of being
translated properly, all mRNAs were translated in a rabbit reticulocyte lysate (Promega) and analyzed on an SDS-12%
polyacrylamide gel.
Culture conditions and embryo manipulations.
Embryos were
obtained by natural fertilization using standard procedures.
Microinjection of capped synthetic RNA was performed as described
before (18). Embryonic stages were determined according to
the method of Nieuwkoop and Faber (15).
Histology, whole-mount immunocytochemistry, and in situ
hybridization.
Embryos were fixed in Smith's fixative (2.5%
acetic acid, 0.5%
K2Cr2O7,
4% formaldehyde), followed by embedding in paraffin, sectioning, and
hematoxylin-eosin staining. Whole-mount in situ hybridization was
performed as described previously (22). As a substrate for
alkaline phosphatase, BM purple AP substrate (Boehringer) was used.
RT-PCR analysis.
Capped RNA was synthesized from the
plasmids as described before (18) and injected into the
animal poles of two-cell embryos. Ectoderm explants were isolated from
blastulae and subjected to reverse transcription (RT)-PCR at neurula
stages. Primers used in the RT-PCR were as follows: EF1 alpha,
5'-CAGATTGGTGCTGGATATGC-3' and
5'-CACTGCCTTGATGACTCCTA-3'; Muscle Actin,
5'-GCTGACAGAATGCAGAAG-3' and
5'-TTGCTTGGAGGAGTGTGT-3'; Xnot,
5'-ATACATGGTTGGCACTGA-3' and 5'-CTCCTACAGTTCCACATC-3'; XANF,
5'-AGCTTTCACTAGGAGCCAGA-3' and 5'-AGGTCCAAGGCTCTATCA-3'; NCAM,
5'-GCGGGTACCTTCTAATAGTCAC-3' and 5'-GGCTTGGCTGTGGTTCTGAAGG-3'; and NRP-1,
5'-GGGTTTCTTGGAACAAGC-3' and
5'-ACTGTGCAGGAACACAAG-3'.
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RESULTS |
EED interacts with YY1 and the Drosophila PcG
protein Pho.
Evidence has accumulated that PcG proteins operate as
large, multimeric protein complexes (27). Previously, the
existence of two distinct human PcG protein complexes, the HPC-HPH PcG
complex and the EED-EZH PcG complex, was reported (5, 24,
25, 26, 27, 30). The Drosophila PcG protein Pho
shares extensive homology with the vertebrate transcription factor YY1
(1). So far, it is unknown to which class of PcG
proteins Pho or YY1 belongs. To screen for potential
interactions between YY1 and human PcG proteins, we performed
a directed two-hybrid screen, using a panel of human
PcG cDNAs.
A positive interaction was found between YY1 and the EED protein (Fig.
1A), but no interaction was found between
YY1 and the PcG proteins EZH2, HPH1, HPC2, RING1, and Bmi1 (Fig. 1A).
This indicates that YY1 is part of the EED-EZH PcG protein complex. To
define the domains that are responsible for the interaction between YY1
and EED, we subcloned different parts of YY1 and EED in frame with the
GAL4 DNA-binding domain and tested whether these proteins could still
interact with full-length YY1 or full-length EED. YY1 contains a
C-terminal domain that contains a zinc finger binding domain that is
highly conserved between YY1 and Pho (32, 38). This region
is also involved in mediating repression (38). YY1 further
contains a spacer region that is not conserved between YY1 and Pho
(1). We found that EED interacts with the C-terminal region of YY1 (amino acids [aa] 250 to 414), hardly with aa 128 to
250, the region that overlaps the spacer region (aa 198 to 295),
and not at all with the N-terminal region (aa 1 to 135) (Fig. 1B). We
conclude that EED binds to the C-terminal region of YY1, which
encompasses the zinc finger binding domain.
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FIG. 1.
Interaction between the EED and YY1 proteins. (A)
Two-hybrid analysis with YY1 and the indicated vertebrate PcG proteins
showed a positive interaction between YY1 and EED. The PcG
cDNAs were cloned in frame with the GAL4 DNA-binding domain
(DBD), and the YY1 cDNA was cloned in frame with the GAL4
transactivation domain (AD). No interactions between the PcG proteins
and simian virus 40 were observed. (B) The domain of YY1 that
interacted with EED was mapped. Indicated portions of YY1 were fused to
the GAL4 AD. -Gal, -galactosidase. (C) Deletion of the most
N-terminal WD-40 domain in EED resulted in abolishment of the
interaction between YY1 and EED.
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EED contains five WD-40 domains, and it was previously found that all
WD-40 domains of EED are necessary for the interaction
between EED and
EZH2 (
30). A truncated EED protein that still
contains the
most N-terminal four WD-40 domains (aa 1 to 501)
already failed to
interact with YY1 (Fig.
1C). It is therefore
probable that, as for the
interaction between EED and EZH2, all
five WD-40 domains of EED need to
be present for a proper interaction
between YY1 and
EED.
To test whether the YY1-EED interaction occurs in vivo,
coimmunoprecipitation experiments were performed using antibodies
against the YY1 and EED proteins. Extracts from Ramos cells in
which
EED and EZH2 are expressed at a high level were used (
41).
We detected both EED and EZH2 in the YY1 IP (Fig.
2). Conversely,
YY1 was present in both
the EED and EZH2 IPs (Fig.
2). Previously
it was shown that HPC2 is not
present in EED or EZH2 IPs (26)
(Fig.
2). We have now found that HPC2
was also not present in
the YY1 IP. Conversely, YY1, EED, and EZH2 were
not present in
the HPC2 IP (Fig.
2). Finally, no antigens were detected
when
the specific IP antibodies were replaced by preimmune sera (Fig.
2, mock IP), underlining the specificity of the IPs. Also, when
antibodies against the human PcG proteins BMI-1 and RING1 were
used
instead of anti-HPC2, we did not observe YY1 in these IPs
(data not
shown). These data indicate that, in vivo, YY1 is associated
with the
EED-EZH protein complex but not with the HPC-HPH PcG
complex.
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FIG. 2.
In vivo interaction between EED and YY1 or Pho. Extracts
from human Ramos cells were immunoprecipitated using antibodies against
EED, EZH2, YY1, or HPC2 or without antibody (mock IP). Western blots of
the IPs were probed with antibodies against EED, EZH2, YY1, or HPC2. No
antigens were detected when the specific IP antibodies were omitted
from the IPs (mock IP). Input, extract from Ramos cells.
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To determine whether the interaction between EED and YY1 is a direct
interaction, we employed an in vitro pull-down assay.
Previously
described fusions of GST and EED, EZH2, and HPC2 (
41)
were
immobilized to GST-Sepharose and incubated with
[
35S]methionine-labeled, in vitro-translated
YY1. After extensive
washing, the
[
35S]methionine-labeled proteins were analyzed
by SDS-polyacrylamide
gel electrophoresis. The YY1 protein was able to
bind to immobilized
GST-EED but not to GST-EZH2, GST-HPC2, GST-RING1,
or immobilized
GST alone (Fig.
3). We
also tested whether the
Drosophila PcG
protein Pho
interacted with GST-EED. As shown in Fig.
3, Pho also
bound to
immobilized GST-EED but not to GST-EZH2, GST-HPC2, GST-RING1,
or
immobilized GST alone. These data confirm the two-hybrid data
and the
coimmunoprecipitation experiments and indicate that YY1
and Pho
interact directly with the EED protein but not with EZH2,
HPC2, or
RING1.
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FIG. 3.
In vitro interaction between EED and YY1 or Pho. In
vitro-translated, [35S]methionine-labeled YY1 or Pho was
incubated with immobilized GST [GST ( )], GST-EED, GST-EZH2,
GST-HPC2, or GST-RING1. The input was 15% of the amount that was
incubated with the GST fusion proteins.
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Identification and characterization of Xeed, a
Xenopus homolog of the PcG protein EED.
In order to
test the functional significance of the EED-YY1 interaction, we wished
to modify the expression levels of these proteins in Xenopus
embryos. As a first step towards this goal, we needed to isolate
Xenopus homologs of YY1 and EED. Since the Xenopus homolog of YY1 has been described previously
(21), we concentrated our efforts on the isolation of the
Xenopus homolog of EED. We screened a Xenopus
oocyte cDNA library with a probe which contains the coding region
of EED (30) and obtained several positive clones. The
longest, a 1,324-bp cDNA clone, was further characterized (Fig.
4). Sequence analysis revealed a
1,278-bp open reading frame. A stop codon was found at the 3'
end of the clone, corresponding with a similar stop codon in the
EED cDNA. Several stop codons were present approximately 15 bp
upstream from the first ATG (data not shown). These data indicate that we isolated a full-length cDNA. Within the 1,278-bp coding region, the cDNA is 81% identical to EED at the nucleotide level. At the protein level the isolated cDNA is 92% identical and 96% similar to the EED protein (30) (Fig. 4). We therefore conclude
that we isolated a Xenopus homolog of EED, which we call
Xeed.
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FIG. 4.
Predicted amino acid sequence of Xeed. The Xeed protein
was compared with the human EED protein. Identical amino acids are
shaded. Five WD-40 repeats are indicated with boxes.
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We characterized the temporal expression of the Xeed and XYY1 genes
through early embryonic development of
Xenopus. Total
RNA
from several developmental stages was analyzed by Northern
blot
analysis. We detected 3- and 4-kb transcripts for the Xeed
gene and an
approximately 5.5-kb single transcript for the XYY1
gene (Fig.
5). The highest expression level of both
transcripts
is found in the fertilized egg throughout blastula stages.
These
transcripts are of maternal origin, since transcription in the
embryo is activated only after the midblastula transition. The
abundance of the Xeed transcript declines during gastrula stages,
and
the abundance of the XYY1 transcript declines during neurula
stages.
However, transcription of both Xeed and XYY1 was observed
throughout
development, from neurula stages onward, indicating
zygotic
transcription. We also examined the spatial distribution
of the Xeed
and XYY1 transcripts in late neurula and tailbud stages
by in situ
hybridization. Expression was detected in tissues such
as the
developing neural tube (data not shown). The analysis did
not reveal,
however, a strongly localized expression of the transcripts
in
the early embryos (data not shown).
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FIG. 5.
Developmental expression profiles of Xeed and XYY1.
Probes for the Xeed and XYY1 genes were used for Northern analysis of
total RNA (20 µg) isolated from the indicated developmental stages
(15). The filter was rehybridized with a probe for G s-1
(19) to verify the loading and integrity of RNA in each
lane.
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In summary, we have isolated a highly conserved
Xenopus
homolog of EED, Xeed. Xeed and XYY1 have similar temporal expression
profiles, and they have a strong maternal component of
expression.
Overexpression of Xeed, XYY1, and Pho induces an ectopic neural
tube.
In order to test whether interference with the expression
levels of Xeed and XYY1 proteins might influence early embryonic development, we injected Xeed or XYY1 mRNA into one blastomere of
two-cell Xenopus embryos. Blastopore formation during
gastrulation and the formation of the neural plate proceeded normally
in both Xeed- and XYY1-injected embryos. However, a broadening
of the neural plate at the injected side of the embryo became
apparent at late neurula and tailbud stages (Fig.
6C and E). When 1 ng of either Xeed or
XYY1 mRNA was injected, this phenotype was observed in
approximately 30% (n = 251) of the embryos.
Importantly, also when 1 ng of Pho mRNA was injected, the same
phenotype was observed and with a similar frequency (data
not shown). Coinjection of trace amounts of -galactosidase
mRNA showed that the broadening of the neural plate had occurred in
the injected side of the embryo (data not shown). Next, we
characterized the embryos by histology. This analysis revealed an
ectopic neural axis at the injected side of the embryo (Fig. 6D and F).
Importantly, we observed neither a notochord underlying the ectopic
neural axis nor somites adjacent to the ectopic neural axis in any of
the embryos we analyzed (Fig. 6D and F).
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FIG. 6.
Phenotypes resulting from injection of Xeed, XYY1, and
XYY1-EnR mRNA into Xenopus embryos. (A,
C, E, and G) Indicated mRNAs were injected into one blastomere of
two-cell-stage Xenopus embryos. The side of the embryo
that was injected is indicated. The broadening of the neural plate in
Xeed-injected (C) and XYY1-injected (E) embryos, compared to the
uninjected embryo (A), is indicated with brackets. The ectopic axis in
XYY1-EnR-injected embryos (G) is also indicated, as is the
cement gland (C). (B, D, F, and H) The embryos in panels A, C, E, and G
were processed for histological analysis in order to visualize the
somites, notochord, and neural tissue. Shown are histological sections
of uninjected (B), Xeed-injected (D), XYY1-injected (F), and
XYY1-EnR-injected (H) embryos.
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EED is as a transcriptional repressor (
26). YY1 can also
act as a transcriptional repressor, dependent on the promoter context
(
32). To test whether in
Xenopus embryos XYY1
operates as a
transcriptional repressor, we took advantage of the fact
that
XYY1 has a DNA-binding domain that acts independently of the
domain
that is involved in transcriptional regulation. We fused the
DNA-binding
domain of XYY1 to the Engrailed repressor
(En
R) domain to create a strong transcriptional
repressor (
2).
The resulting
XYY1-En
R mRNA was injected into one
blastomere of two-cell
Xenopus embryos.
We observed the same
phenotype as with either Xeed, XYY1, or Pho,
but with much higher
efficiency. Injection of 50 pg of XYY1-En
R
mRNA (instead of 1 ng of XYY1 mRNA) resulted in broadened
neural
plates in over 50% (
n = 276) of the
injected embryos. A significant
proportion (approximately 15%) of
these embryos displayed a bifurcated
axis (Fig.
6G). This event was
observed only rarely in Xeed- or
XYY1-injected embryos.
Histological analysis of these bifurcated
embryos demonstrated a
secondary, ectopic neural axis in the ventral
regions of the
XYY1-En
R-injected embryos (Fig.
6H). These
secondary axes did not, however,
develop into complete head structures
(Fig.
6H). Importantly,
as in Xeed- and XYY1-injected embryos, no
notochord or somites
were observed in the vicinity of the ectopic
neural axis (Fig.
6H). Given the similarity of the phenotypes and the
fact that
the XYY1-En
R fusion protein is a strong
transcriptional repressor (by virtue
of the En
R
domain), we conclude that the XYY1 protein also acts as a
transcriptional
repressor in
Xenopus embryos.
In control experiments we injected mRNAs encoding the
Xenopus PcG proteins XPc (
22) and Xbmi1
(
22) or the RING1 protein
(
24) into one cell
of a two-cell-stage embryo. We observed neither
phenotypic effects nor
changes at the histological level when
XPc (Fig.
7A and B) was injected. No effects were
observed with
either Xbmi1 or RING1 (data not shown). Also, mutants of
Xeed
and XYY1 were injected. The Xeed (aa 1 to 385) mutant consisted
of
Xeed protein in which the most C-terminal WD-40 domain was
removed. The
corresponding EED mutant failed to interact with
YY1 in the two-hybrid
assay (Fig.
1C). The XYY1 mutant (aa 1 to
250) consisted of XYY1
protein from which the C-terminal, zinc
finger binding domain was
removed. The corresponding C-terminal
region of YY1 was identified as
mediating the binding to EED (Fig.
1B). Neither Xeed (aa 1 to 385)
(Fig.
7C and D) nor XYY1 (aa 1
to 250) (Fig.
7E and F) induced
phenotypic changes or changes
at the histological level. Taken
together, these results underline
the specificity of the effects of
Xeed and XYY1 on induction of
neural tissue as well as underline the
functional significance
of the interaction between EED and YY1.
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FIG. 7.
Lack of phenotypes from injection of XPc and mutants of
Xeed and XYY1. Indicated mRNAs were injected into one blastomere of
two-cell-stage Xenopus embryos. The side of the embryo
that was injected is indicated. Injection of neither XPc (A and B),
Xeed (aa 1 to 385) (C and D), nor XYY1 (aa 1 to 250) (D and E) induced
broadening of the neural plate or histologically defined neural
tissue.
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To further study the molecular characteristics of the axis that can be
induced by Xeed and XYY1, we performed in situ hybridization
on Xeed-
and XYY1-injected embryos, using probes against neural
marker genes. We
chose the general neural marker NRP-1, which
is expressed in
the developing neural tube of stage 23 embryos
(
12) (Fig.
8A). We found that the ectopic tissue in
Xeed- and
XYY1-injected embryos was characterized by expression of
NRP-1
(Fig.
8B and C). Also, in XYY1-En
R-injected
embryos, NRP-1 expression in the ectopic axis was observed
(Fig.
8D).
These data confirm the histological analysis and demonstrate
that Xeed
and XYY1 induce neural tissue in the injection site.
We therefore
conclude that overexpression of Xeed and XYY1 induces
a secondary,
ectopic neural axis, but no notochord or somites.
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FIG. 8.
Induction of neural tissue in Xeed- and XYY1-injected
embryos. Xeed (B), XYY1 (C), and XYY1-EnR (D) mRNAs
were injected into one blastomere of the two-cell-stage
Xenopus embryo and cultured until stage 23. All injected
embryos, as well as the uninjected control embryos (A), were subjected
to whole-mount in situ hybridization using a probe that detects the
neural marker NRP-1. Subsequently, the embryos were processed for
histological analysis and sections were examined. The ectopic neural
tissue in Xeed-, XYY1-, and XYY1-EnR-injected embryos is
indicated with arrows.
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Xeed, Pho, XYY1, and XYY1-EnR directly induce neural
tissue in ectoderm explants.
The lack of notochord or somites in
the proximity of the ectopic neural axis indicated that this ectopic
neural tube was the result of a direct neuralizing event. Therefore, we
tested whether Xeed, Pho, XYY1, and XYY1-EnR are
able to directly induce neural tissue in ectoderm tissue. We injected
the respective mRNAs into the animal zone of both blastomeres of
two-cell Xenopus embryos. When these embryos had reached
early gastrula stage (stage 10), we dissected the ectoderm and cultured
them until control embryos had reached the tailbud stage (stage 25).
Total RNA was isolated, and the expression levels of the general neural
markers NRP-1 (12) and NCAM (11) and the
anterior neural marker XANF (45) were examined using
RT-PCR. As shown in Fig. 9A, 2 ng of Xeed
mRNA (lane 4), 2 ng of XYY1 mRNA (lane 5), 2 ng of Pho mRNA
(lane 6), and 100 pg of XYY1-EnR mRNA (lane
7) all induced expression of the NRP-1, NCAM, and XANF neural marker
genes. No expression of these neural markers was induced in control
ectoderm that had been excised from early gastrula ectoderm (lane 3)
and subsequently cultured to the tailbud stage. No neural markers were
induced in cultured ectoderm of embryos which were injected with either
2 ng of XPc mRNA (lane 8) or 2 ng of XBmi1 mRNA (data not
shown).
View larger version (49K):
[in this window]
[in a new window]
|
FIG. 9.
Direct neural induction in ectoderm by Xeed and XYY1.
(A) Two nanograms of Xeed, XYY1, or Pho mRNA, 100 pg of
XYY1-EnR mRNA, and 2 ng of XPc mRNA were injected
into two blastomeres of two-cell Xenopus embryos and
cultured to stage 10 (early gastrula). The entire ectoderm was
dissected and cultured to stage 25, when RNA was isolated and analyzed
by RT-PCR using primers recognizing the neural markers NRP-1, NCAM, and
XANF as well as the mesodermal marker -actin and the notochord
marker Xnot. As a control, dissected stage 10 ectoderm of an uninjected
embryo (lane 3) was cultured to stage 25. Uninjected, whole stage 23 embryos were analyzed as a positive control (lane 1). To verify the
presence of equal amounts of RNA, EF1 was used as loading
marker. RT, no reverse transcriptase was added. (B) Two
hundred picograms of Xeed (lane 3), 200 pg of XYY1 (lane 4), or 200 pg
of Xeed plus 200 pg of XYY1 (lane 5) were injected into two blastomeres
of two-cell Xenopus embryos and the experiment proceeded
as for panel A.
|
|
We also determined whether there had been induction of mesodermal
markers in the cultured ectoderm explants of the injected
embryos. We
monitored the expression levels of the mesodermal
somite marker
-actin (
13) and the notochord marker Xnot
(
40).
No expression of these markers was found in cultured
control ectoderm
explants (Fig.
9A, lane 3) or in cultured explants of
embryos
injected with Xeed (lane 4), XYY1 (lane 5), Pho (lane 6),
XYY1-En
R (lane 7), or XPc (lane 8)
mRNA.
We also tested whether coinjected Xeed and XYY1 might synergize to
induce neural tissue. When 200 pg of either Xeed (Fig.
9B, lane 3) or
XYY1 (Fig.
9B, lane 4) mRNA was injected instead
of 2 ng of
mRNA (Fig.
9A), low expression levels of NRP-1 and
XANF were
induced. However, coinjection of 200 pg of Xeed mRNA
plus 200 pg of
XYY1 mRNA (Fig.
9B, lane 5) induced strong expression
of NRP-1 and
XANF, indicating that Xeed and XYY1 synergize to
induce neural
tissue.
These data confirm the histological analysis showing that Xeed, Pho,
XYY1, and XYY1-En
R are able to induce ectopic
neural tissue that is not accompanied
by somite or notochord tissue.
Importantly, this implies that
the effects of these genes are due to
direct neural induction
within the ectoderm and are not a secondary
event resulting from
initial mesoderm
induction.
| |
DISCUSSION |
YY1 interacts with the PcG protein EED but not with other PcG
proteins.
The Drosophila Pho protein (1)
is, together with mel-18 (9), the only known PcG protein
that displays DNA-binding properties. Since all other known PcG
proteins lack this ability, this is a very important feature. It is,
therefore, important to know to which PcG complex the Pho protein, or
its vertebrate homolog, YY1, belongs. Potentially, YY1 could be part of
either the HPC-HPH or the EED-EZH PcG protein complex. Hence, we used a
panel of cDNAs cloned into two-hybrid vectors to investigate if and
to which known human PcG protein YY1 binds. We show that YY1 interacts specifically with EED, the PcG protein that forms a complex with EZH2
(30) and HDAC proteins (41). We substantiated
the two-hybrid interaction between EED and YY1 by performing in vivo
coimmunoprecipitations and in vitro GST pull-down studies. Using both
methods we demonstrated the validity of the two-hybrid interaction
between EED and YY1. The identification of the interaction between EED
and YY1 allows us to extend a previous model of human PcG protein
complexes (27). In Fig. 10
we show our current model, in which YY1 is part of the complex that
encompasses the vertebrate PcG proteins EED and EZH2. It is important
to note that our present findings are consistent with the fact that
HDAC activity is associated with the EED-EZH complex and not with the
HPC-HPH complex (36, 41). Since it has previously been
demonstrated that YY1 interacts with HDAC proteins (44),
it also implies that HDAC proteins have two possibilities to interact
with the EED-EZH complex: with EED (41) and with YY1
(44; also this work).
View larger version (29K):
[in this window]
[in a new window]
|
FIG. 10.
Composition of human PcG protein complexes. The HPC-HPH
PcG complex contains the PcG proteins HPC2, BMI1, HPH1, HPH2, and
RING1A. The EED-EZH PcG complex contains the PcG proteins EED and EZH2
and is associated with HDAC proteins. In this article we show that the
homolog of the Drosophila PcG protein Pho, YY1,
interacts with the EED protein and thus is either part of or associated
with the EED-EZH PcG complex.
|
|
EED is a vertebrate homolog of the
Drosophila PcG protein
esc (
6,
37). esc is distinguished from other PcG proteins
in
Drosophila in that it is primarily required only during
embryogenesis.
In a previous paper it was speculated that deacetylation
of histones
by HDACs and the recruitment of EED to the HDAC proteins
may be
among the initial repressive events during embryogenesis that
eventually lead to stable and heritable PcG-mediated repression
of
target genes (
41). Now we have found that YY1 is part of
or associated with the EED-EZH PcG complex, which displays HDAC
activity. Since Pho and YY1 display specific DNA-binding properties,
this finding suggests a model in which YY1 directs the EED-EZH
PcG
complex to target genes (Fig.
10). This first step is consistent
with
the early developmental role of the EED-EZH complex, as has
been
defined genetically. It is also consistent with a role for
histone
deacetylation, mediated by the HDACs, which are associated
with both
EED and YY1, as an early event by which PcG proteins
set up stable
repression of target
genes.
The existence of two distinct PcG protein complexes has also been
observed in
Drosophila. Using a two-hybrid analysis, the
ESC and E(Z) proteins have been found to interact (
8,
39).
Furthermore, two distinct
Drosophila PcG protein
complexes have
been characterized biochemically. One complex contains
the PC,
PSC, and PH proteins (
31); the other contains the
ESC and E(Z)
proteins (
14). These findings are very
similar to the observations
in the human system (
30,
41).
There are, however, significant
differences between the two
developmental systems. For instance,
the
Drosophila Pho
protein lacks a domain that mediates histone
deacetylation activity
(
1). This domain is present in the YY1
protein. It is
possible that this constitutes a fundamental difference
between the
Drosophila and the vertebrate systems, indicating
that
histone deacetylation plays a less significant role in the
Drosophila system. Further, Shao and coworkers did not
detect
the Pho protein in the PC-PH complex (
31), which is
in agreement
with our present findings. However, the Pho protein could
not
be detected in the biochemically purified ESC-E(Z) complex either
(
14). These puzzling findings may reflect a more
transient nature
of interactions between Pho and other proteins,
which precludes
biochemical purification as part of a stable
protein complex.
Our observation that even an in vitro interaction
between EED
and the
Drosophila Pho protein exists at least
suggests a highly
conserved nature of the interaction between EED and
YY1. Also,
the virtually identical phenotypes that are induced by Xeed,
XYY1,
and Pho in
Xenopus embryos suggest that YY1 or Pho is
either a
stable component of or at least transiently associated with
the
EED-EZH PcG complex and not the HPC-HPH PcG
complex.
Role of Xeed and XYY1 in the induction of neural tissue in
Xenopus.
To study the functional significance of
the interaction between EED and YY1, we manipulated the expression
levels of the Xenopus homologs of these proteins, Xeed and
XYY1. Both proteins, but no other PcG proteins, induced an ectopic
neural axis in Xenopus embryos, but neither Xeed nor XYY1
was able to induce mesodermal tissue, such as muscle or notochord.
Importantly, the Drosophila Pho protein induced the same
phenotype. The similarity of effects underlines the significance of the
EED-YY1 interaction. The fact that Pho induced the same phenotype and
neural tissue in ectoderm explants also substantiates the notion that
YY1 is indeed a functional homolog of the Drosophila Pho protein.
Our data point towards an early developmental role for the EED-EZH
complex. Also, in homozygous
eed/ mice
the earliest developmental decisions are affected, pointing
towards an
early role for EED in setting up vertebrate PcG-mediated
repression. It
may be significant that homozygous
eed/
mice lack a node and, probably as a consequence of this, also
neural
tissue (
28). Whereas the homozygous
YY1/ mutation is embryonic lethal, in
heterozygote
YY1/+ mice the formation of
a proper neural tube is seriously hampered
(
3). Both
phenotypes are complementary to the phenotypes we
observed after
overexpression of both Xeed and XYY1 proteins in
Xenopus
embryos. Although a detailed comparison between the
loss-of-function
data in mice and overexpression of proteins in
Xenopus is not
possible, the opposing effects on neural
tissue are compatible
with each other. The results reinforce one
another and both point
towards an early role of these PcG proteins in
developmental decisions,
such as the induction of embryonic
tissues.
How do our findings relate to other neural inducing factors that have
been identified over the past few years? Most knowledge
concerning
pathways that mediate the earliest induction steps
has been linked to
secreted proteins. A common theme emerges indicating
that neural
induction results from antagonizing the bone morphogenetic
protein signaling pathway (
7,
43). Secreted
proteins, such
as noggin (
34), are able to directly induce
neural tissue in
Xenopus ectoderm by antagonizing bone
morphogenetic protein signaling.
Further, overexpression of proteins
involved in conserved signal
transduction pathways, such as protein
kinase C, enhances the
competence of ectoderm to become induced to
neural tissue, but
these proteins lack the ability to directly convert
ectoderm into
neural tissue (
16,
17,
18). In that respect,
the involvement
of protein kinase C in mediating neural competence
resembles the
role of another chromatin-associated factor. Recently it
has been
found that overexpression of histone H1 limits the ability of
Xenopus ectoderm to become mesoderm (
35).
Interference with
histone H1 expression does not directly induce
mesoderm but changes
the time window in which ectoderm can be induced
to become mesoderm
(
35). Beside histone H1, no involvement
of chromatin-associated
factors in mediating embryonic induction
phenomena has been described.
We therefore believe that our data reveal
a novel type of factors
that are involved in
Xenopus neural
induction.
The following questions remain: which are the target genes of Xeed and
XYY1, and how does the modulation of the activity of
these target genes
result in the induction of neural tissue? Since
EED is a repressor of
gene activity (
30), it is likely that
Xeed is also a
repressor of gene activity. Furthermore, both XYY1
and
XYY1-En
R directly induce neural tissue, and by
virtue of the En
R domain,
XYY1-En
R is a transcriptional repressor. It is,
therefore, likely that
the target genes of Xeed and XYY1 are repressed
by these proteins
and that this repression results in the induction of
neural tissue.
It will be of considerable interest to identify these
target genes.
Since the effects of Xeed and XYY1 occur early in
development,
these target genes may well represent a class of PcG
target genes
other than the known PcG target genes in
Drosophila that are affected
relatively late during
development. Also, identification of such
target genes may reveal
pathways, distinct from the known ones,
that are involved in mediating
neural induction in
Xenopus.
| |
ACKNOWLEDGMENTS |
We gratefully thank Eddy de Robertis, Peter Good, Richard
Harland, Ali Hemmati-Brivanlou, David Kimelman, Paul Krieg, Doug Melton, Tim Mohun, Ralph Rupp, Colin Sharp, Jim Smith, and
A. G. Zaraisky for providing us with various reagents. We further thank Thijs Hendrix for excellent frog care, Johan van der Vlag for performing the coimmunoprecipitations, Frank
Raaphorst for photography, and Ralph Rupp for stimulating discussions.
This work was sponsored in part by the Human Frontier Science Program
(RG0039/1999-M).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Swammerdam
Institute for Life Sciences, BioCentrum Amsterdam, University of
Amsterdam, Plantage Muidergracht 12, 1018 TV Amsterdam, The
Netherlands. Phone: 31-20-5255115. Fax: 31-20-5255090. E-mail:
arie.otte{at}chem.uva.nl.
Present address: Department of Biology, University of California,
San Diego, La Jolla, CA 92093.
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A novel homeobox gene expressed in the anterior neural plate of the Xenopus embryo.
Dev. Biol.
152:373-382[CrossRef][Medline].
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Molecular and Cellular Biology, February 2001, p. 1360-1369, Vol. 21, No. 4
0270-7306/01/$04.00+0 DOI: 10.1128/MCB.21.4.1360-1369.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
