1 Division of Developmental Biology, Department of Cell and Molecular Biology
and Center for Bioenvironmental Research, Tulane University, New Orleans, LA
70118, USA
2 College of Bioengineering, Fujian Normal University, Fuzhou, Fujian Province,
350007, P. R. China
* Author for correspondence (e-mail: ychen{at}tulane.edu)
Accepted 18 May 2004
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Polycomblike 2 gene, Transcriptional repressor, Left-right asymmetry, Shh, Chick embryo
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
During embryonic development, once cell fate is specified, cell identity is
maintained by epigenetic functions. The Trithorax-group (Trx-G) and
Polycomb group (PcG) genes are part of the widely conserved cell
memory system that maintains both the active and silenced states of
transcription patterns (Kennison,
1995). The PcG proteins are encoded by about 40 genes in
Drosophila, which include Polycomb, Polyhomeotic,
Polycomblike (Pcl) and posterior sex comb. PcG mutants
exhibit posterior homeotic transformation because of the ectopic expression of
the HOM-C genes. Structural homologs of the Drosophila PcG
proteins have been identified in mammals, and the mechanisms by which these
proteins silence target genes and stabilize developmental decisions are likely
to be conserved between Drosophila and vertebrates. Similar to
Drosophila, mutations in different mammalian PcG genes cause
posterior axial transformations of the mouse skeleton and anterior shifts of
Hox expression boundaries (Akasaka et al.,
1996
; Schumacher et al.,
1996
; Coré et al.,
1997
; Takihara et al.,
1997
; Suzuki et al.,
2002
). In addition to the regulation of Hox gene
expression, PcG gene members have been shown recently to regulate
developmentally important genes, such as hedgehog
(Maurange and Paro, 2002
), and
cell cycle regulation genes, such as Rb
(Dahiya et al., 2001
). In
Drosophila, Polycomblike (Pcl), a member of the PcG, plays
an important and perhaps central role in PcG function
(Landecker et al., 1994
). The
Pcl protein contains two Cys4-His-Cys3 motifs, known as
the plant homeodomain (PHD) type zinc finger
(Aasland et al., 1995
). The PHD
motif, also called the leukemia-associated protein domain, is found in more
than 400 eukaryotic proteins. Most of the PHD domain proteins are thought to
be involved in transcription regulation, possibly acting through chromatin
remodeling and histone acetylation (Aasland
et al., 1995
; Yochum and Ayer,
2001
; Kalkhoven et al.,
2002
). More specifically the PHD finger appears to act as a
protein-protein interaction domain to mediate the regulation of gene
expression (Jacobson and Pillus,
1999
). Although the function of the Pcl genes in vertebrate
development remains unclear, studies in Xenopus indicate that they
negatively regulate, or repress, gene expression in the developing anterior
central nervous system (Yoshitake et al.,
1999
; Kitaguchi et al.,
2001
).
In this report, we show that a novel chick Polycomblike gene, chick Pcl2, encodes a transcription repressor and exhibits asymmetric expression in the right side of Hensen's node. Protein-soaked bead implantation studies indicate that chick Plc2 resides downstream of Activin-ßB and Bmp4 in the node. Inhibition of chick Pcl2 expression in the early embryos led to randomization of cardiac looping direction. Using gain-of-function and loss-of-function approaches, we demonstrate that chick Pcl2 is both necessary and sufficient for the repression of Shh expression in the node. The repression of Shh expression by chick Pcl2 seems to be conserved in other developing organs and even across species. Overexpression of chick Pcl2 by RCAS retroviral infection in the developing chick limb bud and feather bud inhibited Shh expression in the ZPA of the limb bud and the epithelia of the feather bud. Transgenic overexpression of chick Pcl2 in the mouse limb bud also inhibited Shh expression in the ZPA. We further demonstrated that chick Pcl2 can repress the activities of the mouse Shh promoter in cell culture assays. Pull-down assays indicate that chick Pcl2 might function as a repressor by recruiting EZH2 via its PHD finger domain. These results indicate that chick Pcl2 plays an essential role in the LR axis specification by silencing Shh expression in the node.
![]() |
Materials and methods |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Probes and in situ hybridization
For the detection of chick Pcl2 expression, the full-length chick
Pcl2 containing plasmid was linearized with XhoI.
Non-radioactive RNA probes were generated and labeled with digoxigenin (DIG)
using T3 RNA Polymerase. Other cDNAs used for gene expression studies include:
a 1.1 kb cDNAs of the chick Nodal
(Levin et al., 1995), a 0.5 kb
Shh cDNA (Ogura et al.,
1996
), a 0.9 kb Caronte cDNA
(Rodriguez-Esteban et al.,
1999
), a 1.8 kb Pitx2 cDNA
(St Amand et al., 1998
), a 1.9
kb SnR cDNA (Isaac et al.,
1997
), a 0.8 kb Fgf8 cDNA
(Meyers and Martin, 1999
), a
1.4 kb Lefty1 cDNA (Schlange et
al., 2001
) and a 0.6 kb mouse Shh cDNA
(Echelard et al., 1993
).
DIG-labeled riboprobes were generated according to the manufacturer's
instruction (Boehringer Mannheim, Indianapolis, IN). Whole-mount and section
in situ hybridization analyses were performed as described previously
(St Amand et al., 1998
;
St Amand et al., 2000
).
Briefly, samples were fixed in freshly made 4% paraformaldehyde/PBS at 4°C
overnight. For whole-mount in situ hybridization, samples were bleached with
6% H2O2 prior to dehydration through a graded methanol
series. For section in situ hybridization, samples were dehydrated through a
graded ethanol series, embedded in paraffin wax and sectioned at 10 µm.
Hybridization signals were visualized using the BM purple AP substrate at
4°C.
Bead implantation and oligonucleotide treatment
Bead implantation experiments were performed on stage 4 chick embryos
explanted in New cultures (New,
1955). Briefly, Affigel-Blue beads (BioRad) were soaked in
Activin-A (500 ng/µl in PBS), Follistatin (500 ng/µl in PBS), BMP4 (1
µg/µl in PBS) or 1% BSA in PBS (as a control), and were implanted on the
left or right side of Hensen's node. All proteins were purchased from R&D
Systems (Minneapolis, MN). Embryos were cultured to desired stages and
collected for whole-mount in situ hybridization analyses. Oligonucleotide
treatment was performed on stage 4-6 chick embryos explanted in New culture as
described previously (Isaac et al.,
1997
; Srivastava et al.,
1995
; Yu et al.,
2001
). The sequence for the 20 base antisense oligonuleotide that
targets the first PHD domain of chick Pcl2 is
5'-CTCCTCCTGACATATTGTAC-3'. The sequence for the random control
oligonucleotide is 5'-GACTATCTAGATAGCTACGT-3'. The oligonuleotides
were synthesized as phosphorothioate derivatives and were purified by HPLC
(IDT, Corralville, IA). About 10 µl oligonucleotide mixed with
lipofectAMINE (GibcoBRL) at a concentration of 40 µM was applied onto
cultured embryos constrained by a plastic ring in New cultures. Embryos were
collected at appropriate stages for analysis of gene expression and for
scoring the direction of cardiac looping.
Expression vectors and microelectroporation
We used microelectroporation to transfer plasmid DNA into early chick
embryonic tissues. To generate expression plasmids, the coding region of chick
Pcl2 (amino acids 1-595) was amplified from the full-length chick
Pcl2 cDNA plasmid using Pfu DNA polymerase (Stratagene, La
Jolla, CA) and was cloned into the pMES expression vector in front of
IRES-Gfp-coding sequence. The resulting plasmid, pMES-Pcl2,
expresses both the transgene and Gfp simultaneously under the control
of the chick ß-actin promoter. To perform microelectroporation,
a gold-plated cathode was fixed to the bottom of a 60 cm dish with a thin
layer of Ringer's saline. Stage 4 chick embryos were prepared for New culture
and embryo constrained by a ring was placed onto the cathode. About 1 µl of
expression plasmids (1 µg/µl), mixed with fast green to visualize DNA,
was injected to the target site between the blastoderm and the vitelline
membrane using a glass capillary. A platinum anode (0.5 mm in diameter) was
then placed on the hypoblast of the node. The distance between the two
electrodes was maintained within 2 mm. Electroporation was performed using BTX
electroporator (Electro Square PoratorTM ECM 830 Model, BTX, San Diego,
CA) with five pulses of 5 V for duration of 25 mseconds and intervals of 454
mseconds. Embryos were then placed onto the agar media in New cultures and
were incubated at 38°C to the appropriate stages. Gfp expression
in targeted tissues was monitored before embryos were harvested for gene
expression analysis.
Retroviral construction and infection
RCAS retroviral construction and infection was performed as described
previously (Yu et al., 2001).
To make the RCAS-Pcl2 construct, the coding region of chick
Pcl2 (amino acids 1-595) was amplified from chick Pcl2 cDNA
plasmid and cloned into the Cla12 vector. The insert was then
released by ClaI digestion and cloned into the RCASBP retroviral
vector. Chicken embryonic fibroblast (CEF) cells expressing RCAS-Pcl2
or RCAS-Gfp were pelleted according to a protocol described
previously (Logan and Francis-West,
1999
). To infect the chick developing limb bud, virus-free chick
eggs (CBT farms, Chestertown, MD) were incubated to approximately stage 12.
RCAS-Pcl2-expressing CEF cells were centrifuged briefly and incubated
as a pellet for 2 hours at 37°C to allow tight cell aggregates to form.
Cell pellets were cut into small pieces and were implanted into the right LPM
of the prospective forelimb-forming region of chick embryos as described
previously (Logan and Tabin,
1998
). The infected embryos were then cultured in ovo and
harvested from stage 19 to stage 24 for morphological and gene expression
analyses. For infection of feather buds, dorsal skin tissues from stage 31
chick embryos were dissected in PBS and transferred to Trowell type organ
cultures in DMEM supplemented with 10% fetal calf serum. About 10 µl RCAS
retroviruses were injected with a microcapillary needle at multiple sites in
the dorsal skin explants. The infected explants were incubated at 37°C in
a 5% CO2 atmosphere for 4 days, and were then collected for gene
expression assays.
Transgenic construct and pronucleus injection
Construction of the chick Pcl2 transgenic construct and pronuclear
injection were performed as described previously
(Zhang et al., 2000). Briefly,
the chick Pcl2 full-length cDNA was cloned into the PCI mammalian
expression vector (Promega, Madison, WI). The chick Pcl2 full-length
cDNA flanked by a chimeric intron and the SV40 late Poly(A) sequence was
released by PstI/BamHI, This fragment was cloned downstream
of the 3.6 kb Hoxb6 promoter
(Schughart et al., 1991
). The
orientation of the insert was confirmed by restriction digestion and
sequencing. Preparation of DNA fragments for injection, collection of zygotes
and pronuclear injection was carried out as described previously
(Hogan et al., 1994
;
Zhang et al., 2000
). Embryos
were collected at E10.75 for analysis of transgene expression and Shh
expression. The integration of the Hoxb6-Pcl2 transgene was
determined by PCR using genomic DNA from the head of each embryo. The PCR
primers used are as follows: 5'-TTTTGGTGCAGCAGGTAGAATAGC-3' (upper
primer) and 5'-CTCCCCCTGAACCTGAAACATAAA-3' (lower primer).
In vitro CAT assays
To map the repressive domain of chick Pcl2 protein, the DNA fragments
encoding the N terminus (amino acids 1-329), the C terminus (amino acids
331-595) and the PHD fingers domain (amino acids 103-238) of chick
Pcl2 were amplified from the full-length chick Pcl2 cDNA
plasmid and cloned into the pBXG1 vector that contains the GAL4 DNA-binding
domain under the control of SV40 enhancer/promoter
(Lillie and Green, 1989). The
derived constructs, named GAL4/Pcl2-N, GAL4/Pcl2-C and
GAL4/PHD, were confirmed by sequencing. The reporter
plasmid, pG5tkCAT, contains the chloramphenicol acetyltransferase
(CAT) reporter gene directed by the herpes simplex virus thymidine kinase (TK)
promoter with five GAL4 binding sites upstream of the TATA-box. Transfection
and CAT assays were carried out in cultured P19 cells, as described previously
(Yu et al., 2001
). A
CMV-ß-gal plasmid was included as an internal control for transfection
efficiency. Transfected cells were cultured for 36 hours and then CAT
activities were determined by thin layer chromatography (TLC) and
scintillation counting. Each experiment was repeated at least three times to
ensure consistent results.
A 1 kb and a 3 kb mouse Shh upstream regions, were amplified using EXLTM Taq (Stratagene, La Jolla, CA) from the mouse genomic clone (RPCI RP23 429M20), and cloned as BamHI-XhoI fragments to replace the TK promoter in pG5tkCAT vector, respectively. The 1 kb Shh upstream region did not give reasonable promoter activities (data not shown). Only the plasmid containing the 3 kb Shh upstream region was used in this study. The pShh-CAT plasmids were cotransfected with pMES-Pcl2, pMES-PHD or pMES (as a control) into P19 cells. Transfection efficiency was monitored by inclusion of CMV-ß-gal plasmid. Transfected cells were cultured for 36 hours prior to CAT assays by both TLC and scintillation counting, which were normalized by protein concentrations. Again, each experiment was carried at least three times.
Immunoprecipitation and protein blotting
To make the constructs for co-immunoprecipitation, the chick Pcl2
full-length cDNA and chick Pcl2 PHD finger domains (amino acids
103-238) were cloned in frame into the pIRES-hrGFP-1 vector
(Stratagene, La Jolla, CA), while the mouse EZH2 sequence was amplified by PCR
from the mouse EZH2 cDNA and cloned into the pCMV-Tag 3A vector (Stratagene).
The resultant expression plasmids, pFLAG-Pcl2, pFLAG-PHD and pMyc-EZH2, were
transiently transfected or co-transfected into 293T cells with
LipofectamineTM 2000 (Invitrogen, Carlsbad, CA). Cells were harvested 48
hours after transfection in a lysis buffer consisting of 0.05 M HEPES, 1%
Triton X-100, 0.15 M NaCl, 10% glycerol, 1 mM EDTA, 1.5 mM
MgCl2·6H2O, Trypsin inhibitor 0.01 mg/ml,
aprotinin 0.01 mg/ml, phenylmethanesufonyl fluoride 1 mM, leupeptin 0.02
mg/ml, sodium orthovanadate 1.25 mM and sodium fluoride 0.1 mM. Lysates were
incubated with either a monoclonal anti-Myc antibody (Abcam, Cambridge, MA) or
an anti-FLAG M2 antibody (Stratagene), and then with Protein A Sepharose beads
(Amersham Pharmacia, Piscataway, NJ). After extensive washing, the eluted
proteins were resolved on PAGE gels, and immunoblotted with an anti-FLAG M2
antibody or a monoclonal anti-Myc antibody, and the binding was detected using
the ECLTM plus Western Blotting Detection system (Amersham
Biosciences).
![]() |
Results |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
|
|
Inhibition of chick Pcl2 expression in the early chick embryo results in ectopic Shh expression in the node and randomized heart looping direction.
Our results established that chick Pcl2 is sufficient for
Shh repression in the node and in other developing organs. We further
examined the function of chick Pcl2 in the development of chick LR
axis by a loss-of-function approach using antisense oliogonucleotides
(Srivastava et al., 1995;
Yu et al., 2001
). A 20 base
antisense oligonucleotide targeting to the first PHD domain of chick
Pcl2 and random control oligonucleotides were synthesized as
phosphorothioate derivatives and were purified by HPLC. Stage 4 chick embryos
were treated with oligonucleotides in New cultures as described previously
(Yu et al., 2001
), and were
allowed to develop to stage 11 for an examination of heart looping direction.
In the studies, 46% (12/26) of embryos treated with antisense oligonucleotides
to chick Pcl2 at stage 4 exhibited reversed cardiac looping
(Fig. 5B), while only 8% (1/12)
of embryos treated with control oligonucleotides exhibited a reversed cardiac
looping (Fig. 5A). However,
when antisense oligonucleotides were applied at stage 5, only 39% (7/18) of
embryos showed reverse cardiac looping; and when applied at stage 6, only 14%
(1/7) showed that phenotype. This stage-dependent effect of antisense
oligonucleotides treatment is consistent with the timing of the asymmetrical
chick Pcl2 expression in the node. Our results indicate that chick
Pcl2 plays a crucial role in the LR axis development in the
chick.
|
Chick Pcl2 encodes a transcription repressor that negatively regulates the activity of the mouse Shh promoter
Products of PcG family genes in Drosophila are required for the
epigenetic repression of homeotic genes and other key developmental regulatory
genes such as hedgehog, through direct transcription effects
(Kennison, 1995;
Maschat et al., 1998
;
Maurange and Paro, 2002
). In
addition, overexpression of the Pcl subfamily members has been shown to
repress target gene expression in vertebrates
(Yoshitake et al., 1999
;
Kitaguchi et al., 2001
). We
next performed in vitro assays to map the repression domain of the chick Pcl2
protein. Constructs containing DNA fragments encoding the chick Pcl2
N terminus (amino acids 1-329), C terminus (amino acids 331-595), and the
region encompassing the two PHD finger domains (amino acids 103-238) which
mediates the association of Drosophila Pcl with ESC/E(Z) repression
complexes (O'Connell et al.,
2001
), fused in frame to the 147 amino acid yeast GAL4 DNA-binding
domain were generated. A construct expressing the N terminus of the chick
Pitx2a transcription activator, which was previously shown to have no effect
on reporter gene expression (Yu et al.,
2001
), was included as control. The transcription activity of the
fusion proteins was assessed on the GAL4-responsive pG5tkCAT
reporter. These assays showed that the fused protein containing the PHD finger
domains (GAL4-Pcl2aa103-238) significantly repressed reporter gene
expression (Fig. 6A).
GAL4-Pcl2N which contains the PHD domains also exhibited a repressive effect
on reporter gene expression. The GAL4-Pcl2C had no repressive effect and
neither did the control vector GAL4-Pitx2aN
(Fig. 6A). It was therefore
concluded that chick Pcl2 indeed encodes for a transcription
repressor and the repression is mediated by PHD finger domain.
|
Chick Pcl2 interacts with EZH2 via its PHD fingers
Generally, PcG proteins function by forming protein complexes with other
proteins. It was shown that the PHD fingers from the Drosophila Pcl
protein interact directly with E(Z) (Enhancer of Zeste), a component of the
ESC/E(Z) repressive complex (O'Connell et
al., 2001). This interaction between Pcl and E(Z) is also
conserved in their human homologs
(O'Connell et al., 2001
). To
test whether chick Pcl2 might also interact with EZH2, the vertebrate homolog
of the E(Z) protein, co-immunoprecipitation studies were performed.
FLAG-tagged chick Pcl2 (FLAG-Pcl2) or PHD fingers (amino acids 103-238;
FLAG-PHD) and Myc-tagged mouse EZH2 (Myc-EZH2) were co-expressed in the 293T
cells. As shown in Fig. 7, the
PHD fingers and EZH2 proteins were steadily pulled down by antibodies against
FLAG or Myc reciprocally, confirming an interaction between EZH2 and the PHD
fingers of chick Pcl2. By contrast, chick Pcl2 proteins failed to pull down
EZH2 reciprocally using the same approach
(Fig. 7). This also happened to
the Drosophila Pcl fusion protein which was unable to but its PHD
finger domain could interact with E(Z)
(O'Connell et al., 2001
). This
could be explained by the mask of the PHD finger domain in chick Pcl2 fusion
protein or instability of chick Pcl2-EZH2 association.
|
![]() |
Discussion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Chick Pcl2 is both sufficient and necessary for the repression of Shh in the node
Hensen's node, the chick organizer, has been a focus in the study of the
molecular mechanism that establishes LR asymmetry. The chick organizer shows
striking asymmetries well before the detection of any overt morphological
signs of asymmetry. For example, the morphological asymmetry is apparent at
the chick node as early as stage 4 (Cooke,
1995; Dathe et al.,
2002
). At the molecular level, a number of genes have been found
to be expressed in or near the chick node in an asymmetric fashion. These
include Activin ßB and its receptor cAct-RIIa
(Levin et al., 1995
;
Levin et al., 1997
),
Bmp4 (Monsoro-Burq and Le Douarin, 2001), Fgf8
(Boettger et al., 1999
),
N-Cadherin (Garcia-Castro et al.,
2000
) and chick Mid1
(Granata and Quaderi, 2003
) in
the right side of the node, and Shh
(Levin et al., 1995
) and
Nodal (Levin et al.,
1995
) in or near the left side of the node. Shh is
initially expressed symmetrically in the node and becomes restricted to the
left side of the node at stage 5 by an inferred Activin-like signal, possibly
Activin-ßB. This asymmetrical Shh in Hensen's node is both
necessary and sufficient for the left-sided expression of Nodal in
the LPM which is conserved in vertebrates
(Levin et al., 1995
;
Pagan-Westphal and Tabin,
1998
). Interestingly, asymmetric expression of Shh in the
node is not observed in mice, zebrafish or rabbit
(Levin et al., 1995
;
Fischer et al., 2002
), arguing
against the conserved mechanism of the initial symmetry-breaking events.
However, Shh mutant mice do display a variety of laterality defects
(Tsukui et al., 1999
). It is
known that the antagonistic interactions between Shh and
Bmp4 maintain the restricted Shh expression in the left side
of Hensen's node (Monsoro-Burg et al., 2001). Recently, chick Mid1, a
microtubule-associated ubiquitin ligase, was shown to act upstream of
Bmp4 to mediate the antagonistic interaction (Granata et al., 2003).
It is reasonable to suspect that a putative transcription repressor is
expressed in the right side of node to mediate the repression of Shh
by the Activin-like signals. We identified such repressor, a novel chick
Polycomblike gene, chick Pcl2, which acts downstream of
Activin and Bmp4 and is expressed in the right side of
Hensen's node. In the node region, chick Pcl2 is expressed in the
ectoderm as is Shh but in a complementary pattern. Chick Pcl2 encodes
a transcription repressor which specifically represses the activity of the
Shh promoter. These studies establish chick Pcl2 as a
candidate for the putative transcriptional repressor of Shh in the
node. In support of this hypothesis, ectopic expression of chick Pcl2
in the left side of Hensen's node abolished Shh expression in the
node and blocked the expression of its downstream genes in the LPM and the
midline. Ablating chick Pcl2 expression by an antisense
oligonucleotide approach caused ectopic Shh expression in the right
side of Hensen's node, which in turn randomized heart looping. These data
indicate that chick Pcl2 is both sufficient and necessary for the
repression of Shh in the right side of the node. In the chick, the LR
identity is liable at stage 4, but becomes fixed at stage 5, concurrent with
the right-sided expression of chick Pcl2 in the node. Chick
Pcl2 seems to participate in stabilizing developmental decision that
establish the LR asymmetry.
Repression of Shh by chick Pcl2 is a conserved feature in different organs and species
Shh, when acting as a morphogen, is essential for crucial pathways that
regulate the differentiation and patterning of a number of tissues. In our
studies, overexpression of chick Pcl2 by electroporation to the node
of stage 4 chick embryos repressed Shh expression in the node and
also in the notochord, a node-derived tissue. Retrovirus-mediated ectopic
expression of chick Pcl2 also repressed Shh expression in
the developing chick limb bud and feather bud. Moreover, transgenic
overexpression of chick Pcl2 in the mouse limb bud similarly
downregulated Shh expression. These consistent results indicate that
the repression of Shh by chick Pcl2 may represent a general
regulatory mechanism for controlling Shh expression in different
organs and tissues in the chick and even across species. The repression of
Shh by chick Pcl2 seems to be a specific rather than a
general repressive effect, because chick Pcl2 represses the mouse
Shh promoter activity but not the TK promoter in vitro.
The repressive effect of chick Pcl2 on Shh expression suggests
that the products of PcG genes not only maintain the silenced status of gene
expression involved in long-term developmental decisions, but also function to
regulate/silence the expression of active gene during embryonic development.
This hypothesis is supported by the finding that in Drosophila the
product of Polyhomeotic (ph) exerts negative transcriptional
control on active genes (Randsholt et al.,
2000). Furthermore, the Xenopus Pcl genes were also shown
to repress gene expression in the developing central nervous system
(Yoshitake et al., 1999
;
Kitaguchi et al., 2001
). It
was recently shown that the antagonistic functions of the Polycomb group
complex and Trithorax complex on a Polycomb response element can govern the
transition between the repressed and active status of gene expression
(Poux et al., 2002
).
Mechanism of chick Pcl2 function
Our results demonstrate that chick Pcl2 encodes a transcription
repressor, and its repression domain was mapped to the PHD fingers. We
demonstrated that the PHD fingers of chick Pcl2 can interact directly with
EZH2, a finding that is consistent with previous results showing that PHD
fingers from both Drosophila and human Pcl bind with high specificity
with EZ in ESC/E(Z) complexes (O'Connell
et al., 2001). Drosophila Pcl was also shown to be a
component of ESC/E(Z) complexes, which contains histone deacetylase and
histone methyltransferase activities (Tie
et al., 2003
; Kuzmichev et
al., 2002
; Muller et al.,
2002
). These enzymatic activities contribute to chromatin
remodeling and transcriptional repression
(Zhang and Reinberg, 2001
).
Based on our evidence showing that chick Pcl2 can specifically repress
Shh promoter activity in vitro and Shh expression in
developing organs, we propose that chick Pcl2 may repress Shh by
recruiting the EED/EHZ2 complex [the mammalian homolog of ESC/E(Z)] through
the conserved interaction between the PHD fingers and EZH2. The potential
DNA-binding domain in chick Pcl2 and chick Pcl2 response
element remain to be identified.
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aasland, R., Gibson, T. J. and Stewart, A. F. (1995). The PHD finger, implication for chromatin-mediated transcriptional regulation. Trends Biochem. Sci. 20, 56-59.[CrossRef][Medline]
Akasaka, T., Kanno, M., Balling, R., Mieza, M. A., Taniguchi, M.
and Koseki, H. (1996). A role for mel-18, a Polycomb
group-related vertebrate gene, during the anterioposterior specification of
the axial skeleton. Development
122,1513
-1522.
Boettger, T., Wittler, L. and Kessel, M. (1999). FGF8 functions in the specification of the right body side of the chick. Curr. Biol. 9, 277-280.[CrossRef][Medline]
Campione, M., Steinbeisser, H., Schweickert, A., Deissler, K.,
van Bebber, F., Lowe, L. A., Nowotschin, S., Viebahn, C., Haffter, P., Kuehn,
M. R. et al. (1999). The homeobox gene Pitx2, mediator of
asymmetric left-right signaling in vertebrate heart and gut looping.
Development 126,1225
-1234.
Capdevila, J., Vogan, K. J., Tabin, C. J. and Izpisúa-Belmonte, J. C. (2000). Mechanisms of left-right determination in vertebrates. Cell 101, 9-21.[Medline]
Collignon, J., Varlet, I. and Robertson, E. J. (1996). Relationship between asymmetric nodal expression and the direction of embryonic turning. Nature 381,155 -158.[CrossRef][Medline]
Cooke, J. (1995). Vertebrate embryo handedness. Nature 374,681 .[Medline]
Coré, N., Bel, S., Gaunt, S. J., Aurrand-Lions, M.,
Pearce, J., Fisher, A. and Djabali, M. (1997). Altered
cellular proliferation and mesoderm patterning in Polycomb-M33-deficient mice.
Development 124,721
-729.
Dahiya, A., Wong, S., Gonzalo, S., Gavin, M. and Dean, D. C. (2001). Linking the Rb and polycomb pathways. Mol. Cell. 8,557 -569.[Medline]
Dathe, V., Gamel, A., Manner, J., Brand-Saberi, B. and Christ, B. (2002). Morphological left-right asymmetry of Hensen's node precedes the asymmetric expression of Shh and Fgf8 in the chick embryo. Anat. Embryol. (Berl). 205,343 -354.[CrossRef][Medline]
Echelard, Y., Epstein, D. J., St-Jacques, B., Shen, L., Mohler, J., McMahon, J. A. and McMahon, A. P. (1993). Sonic hedgehog, a member of a family of putative signaling molecules, is implicated in the regulation of CNS polarity. Cell 75,1417 -1430.[Medline]
Fischer, A., Viebahn, C. and Blum, M. (2002). FGF8 acts as a right determinant during establishment of the left-right axis in the rabbit. Curr. Biol. 12,1807 -1816.[CrossRef][Medline]
Garcia-Castro, M. I., Vielmetter, E. and Bronner-Fraser, M.
(2000). N Cadherin, a cell adhesion molecule involved in
establishment of embryonic left-right asymmetry.
Science 288,1047
-1051.
Granata, A. and Quaderi, N. A. (2003). The Opitz syndrome gene MID1 is essential for establishing asymmetric gene expression in Hensen's node. Dev. Biol. 258,397 -405.[CrossRef][Medline]
Hamburger, V. and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,49 -92.
Hemmati-Brivanlou, A., Kelly, O. G. and Melton, D. A. (1994). Follistatin, an antagonist of activin, is expressed in the Spemann organizer and displays direct neuralizing activity. Cell 77,283 -295.[Medline]
Hogan, B., Beddington, R., Constantini, F. and Lacy, E. (1994). Manupulating the Mouse Embryo, A Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Hyatt, B. A., Lohr, J. L. and Yost, H. J. (1996). Initiation of vertebrate left-right axis formation by maternal Vg1. Nature 384, 62-65.[CrossRef][Medline]
Iemura, S., Yamamoto, T. S., Takagi, C., Uchiyama, H., Natsume,
T., Shimasaki, S., Sugino, H. and Ueno, N. (1998). Direct
binding of follistatin to a complex of bone-morphogenetic protein and its
receptor inhibits ventral and epidermal cell fates in early Xenopus embryo.
Proc. Natl. Acad. Sci. USA
95,9337
-9342.
Isaac, A., Sargent, M. G. and Cooke, J. (1997).
Conntrol of vertebrate left-right asymmetry by a snail-related zinc finger
gene. Science 275,1301
-1304.
Jacobson, S. and Pillus, L. (1999). Modifying chromatin and concepts of cancer. Curr. Opin. Genet. Dev. 9,175 -184.[CrossRef][Medline]
Kalkhoven, E., Teunissen, H., Howweling, A., Verrijzer, C. P.
and Zantema, A. (2002). The PHD type zinc finger is an
integral part of the CBP acetyltransferase domain. Mol. Cell.
Biol. 22,1961
-1970.
Kawakami, M. and Nakanishi, N. (2001). The role
of an endogenous PKA inhibitor, PKI-alpha, in organizing left-right axis
formation. Development
128,2509
-2515.
Kennison, J. A. (1995). The Polycomb and trithorax group proteins of Drosophila, trans-regulators of homeotic gene function. Annu. Rev. Genet. 29,289 -303.[CrossRef][Medline]
Kitaguchi, T., Nakata, K., Nagai, T., Aruga, J. and Mikoshiba, K. (2001). Xenopus Polycomblike 2 (XPxl2) controls anterior to posterior patterning of the neural tissue. Dev. Genes Evol. 211,309 -314.[CrossRef][Medline]
Knezevic, V., de Santo, R., Schughart, K., Huffstadt, U.,
Chiang, C., Mahon, K. A. and Mackem, S. (1997). Hoxd-12
differentially affects preaxial and postaxial chondrogenic branches in the
limb and regulates Sonic hedgehog in a positive feedback loop.
Development 124,4523
-4536.
Kuzmichev, A., Nishioka, K., Erdjument-Bromage, H., Tempst, P.
and Reinberg, D. (2002). Histone methyltransferase activity
associated with a human multiprotein complex containing the Enhancer of Zeste
protein. Genes Dev. 16,2893
-2905.
Landecker, H. L., Sinclair, D. A. and Brock, H. W. (1994). Screen for enhancers of Polycomb and Polycomblike in Drosophila melanogaster. Dev. Genet. 15,425 -434.[Medline]
Levin, M., Johnson, R. L., Stern, C. D., Kuehn, M. and Tabin, C. J. (1995). A molecular pathway determining left-right asymmetry in chick embryogenesis. Cell 82,803 -814.[Medline]
Levin, M., Pagan, S., Roberts, D. J., Cooke, J., Kuehn, M. R. and Tabin, C. J. (1997). Left/right patterning signals and the independent regulation of different aspects of situs in the chick embryo. Dev. Biol. 189,57 -67.[CrossRef][Medline]
Lillie, J. W. and Green, M. R. (1989). Transcription activation by the adenovirus E1a protein. Nature 338,39 -44.[CrossRef][Medline]
Logan, C. and Francis-West, P. (1999). Gene transfer in avian embryos using replication-competent retroviruses. In Methods Mol. Biol. (ed. P. T. Sharp and I. Mason), Vol. 97. Totowa, NJ: Humana Press.
Logan, M. and Tabin, C. J. (1998). Targeted gene misexpression in chick limb buds using avian replication-competent retroviruses. Methods 14,407 -420.[CrossRef][Medline]
Logan, M., Pagán-Westphal, S. M., Smith, D. M., Paganessi, L. and Tabin, C. J. (1998). The transcription factor Pitx2 mediates situs-specific morphogenesis in response to left-right asymmetric signals. Cell 94,307 -317.[Medline]
Loots, G., Ovcharenko, I., Pachter, L., Dubchak, I. and Rubin,
E. (2002). rVISTA for comparative sequence-based discovery of
functional transcription factor binding sites. Genome.
Res. 12,832
-839.
Lowe, L. A., Supp, D. M., Sampath, K., Yokoyama, T., Wright, C. V. E., Potter, S. S., Overbeek, P. and Kuehn, M. R. (1996). Conserved left-right asymmetry of nodal expression and alterations in murine situs inversus. Nature 381,158 -161.[CrossRef][Medline]
Maschat, F., Serrano, N., Randsholt, N. B. and Geraud, G.
(1998). Engrailed and polyhomeotic interactions are required to
maintain the A/P boundary of the Drosophila developing wing.
Development 125,2771
-2780.
Maurange, C. and Paro, R. (2002). A cellular
memory module conveys epigenetic inheritance of hedgehog expression during
Drosophila wing imaginal disc development. Genes Dev.
16,2672
-2683.
Meyers, E. N. and Martin, G. R. (1999).
Differences in left-right axis pathways in mouse and chick, Function of FGF8
and SHH. Science 285,403
-406.
Momose, T., Tonegawa, A., Takeuchi, J., Ogawa, H., Umesono, K. and Yasuda, K. (1999). Efficient targeting of gene expression in chick embryo by microelectroporation. Develop. Growth Differ. 41,335 -344.[CrossRef][Medline]
Monsoro-Burg, A. and Le Douarin, N. M. (2001). BMP4 plays a key role in left-right patterning in chick embryos by maintaining Sonic hedgehog asymmetry. Mol. Cell 7, 789-799.[CrossRef][Medline]
Muller, J., Hart, C. M., Francis, N. J., Vargas, M. L., Sengupta, A., Wild, B., Miller, E. L., O'Connor, M. B., Kingston, R. E. and Simon, J. A. (2002). Histone methyltransferase activity of a Drosophila Polycomb group repressor complex. Cell 111,197 -208.[Medline]
New, D. A. T. (1955). A new technique for the cultivation of the chick embryo in vitro. J. Embryol. Exp. Morphol. 3,326 -331.
O'Connell, S., Wang, L., Robert, S., Jones, C. A., Saint, R. and
Jones, R. S. (2001). Polycomblike PHD fingers mediate
conserved interaction with Enhancer of Zeste protein. J. Biol.
Chem. 276,43065
-43073.
Ogura, T., Alvarez, I. S., Vogel, A., Rodriguez, C., Evans, R.
M. and Izpisua Belmonte, J. C. (1996). Evidence that Shh
cooperates with a retinoic acid inducible co-factor to establish ZPA-like
activity. Development
122,537
-542.
Pagan-Westphal, S. M. and Tabin, C. J. (1998). The transfer of left-right positional information during chick embryogenesis. Cell 93,25 -35.[Medline]
Patel, K., Isaac, A. and Cooke, J. (1999). Nodal signalling and the roles of the transcription factors SnR and Pitx2 in vertebrate left-right asymmetry. Curr. Biol. 9, 609-612.[CrossRef][Medline]
Pearse, R. V., II, Vogan, K. J. and Tabin, C. J. (2001). Ptc1 and Ptc2 transcripts provide distinct readouts of hedgehog signaling activity during chick embryogenesis. Dev. Biol. 239,15 -29.[CrossRef][Medline]
Piedra, M. E. and Ros, M. A. (2002). BMP signaling positively regulates Nodal expression during left right specification in the chick embryo. Development 129,3431 -3440.[Medline]
Piedra, M. E., Icardo, J. M., Albajar, M., Rodriguez-Rey, J.-C. and Ros, M. A. (1998). Pitx2 participates in the late phase of the pathway controlling left-right asymmetry. Cell 94,319 -324.[Medline]
Ponting, C. P. (1997). Tudor domains in proteins that interact with RNA. Trends Biochem. Sci. 22, 51-52.[CrossRef][Medline]
Poux, S., Horrad, B., Sigrist, C. J. and Pirrotta, V. (2002). The Drosophila trithorax protein is a coactivator required to prevent re-establishment of polycomb silencing. Development 129,2483 -2493.[Medline]
Randsholt, N. B., Maschat, F. and Santamaria, P. (2000). Polyhomeotic controls engrailed expression and the hedgehog signaling pathway in imaginal discs. Mech. Dev. 95,89 -99.[CrossRef][Medline]
Raya, A., Kawakami, Y., Rodriguez-Esteban, C., Ibanes, M., Rasskin- Gutman, D., Rodriguez-Leo, J., Buscher, D., Feijo, J. and Izpisúa-Belmonte, J. C. (2004). Notch activity acts as a sensor for extracellular calcium during vertebrate left-right determination. Nature 427,121 -128.[CrossRef][Medline]
Riddle, R. D., Johnson, R. L., Laufer, E. and Tabon, C. (1993). Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75,1401 -1416.[Medline]
Rodriguez-Esteban, C., Capdevila, J., Economides, A. N., Pascual, J., Ortiz, A. and Izpisúa-Belmonte, J. C. (1999). The novel Cer-like protein Caronte mediates the establishment of embryonic left-right asymmetry. Nature 401,243 -251.[CrossRef][Medline]
Rodriguez-Esteban, C., Capdevila, J., Kawakami, Y. and Izpisúa-Belmonte, J. C. (2001). Wnt signaling and PKA control Nodal expression and left-right determination in the chick embryo. Development 128,3189 -3195.[Medline]
Ryan, A. K., Blumberg, B., Rodriguez-Esteban, C., Yonei-Tamura, S., Tamura, K., Tsukui, T., de la Pena, J., Sabbagh, W., Greenwald, J., Choe, S. et al. (1998). Pitx2 determines left-right asymmetry of internal organs in vertebrates. Nature 394,545 -551.[CrossRef][Medline]
Sampath, K., Cheng, A. M. S., Frisch, A. and Wright, C. V.
E. (1997). Functional differences among Xenopus nodal-related
genes in left-right axis determination. Development
124,3293
-3302.
Schlange, T., Schnipkoweit, I., Andree, B., Ebert, A., Zile, M. H., Arnold, H. H. and Brand, T. (2001). Chick cfc controls lefty1 expression in the embryonic midline and nodal expression in the lateral plate. Dev. Biol. 234,376 -389.[CrossRef][Medline]
Schlange, T., Arnold, H. H. and Brand, T. (2002). BMP2 is a positive regulator of Nodal signaling during left-right axis formation in the chick embryo. Development 129,3421 -3429.[Medline]
Schneider, A., Mijalski, T., Schlange, T., Dai, W., Overbeek, P., Arnold, H. H. and Brand, T. (1999). The homeobox gene Nkx3.2 is a target of left-right signaling and is expressed on opposite sides in chick and mouse embryos. Curr. Biol. 9, 911-914.[CrossRef][Medline]
Schumacher, A., Faust, C. and Magnuson, T. (1996). Positional cloning of a global regulator of anterior-posterior patterning in mice. Nature 383,250 -253.[CrossRef][Medline]
Schughart, K., Bieberich, C. J., Eid, R. and Ruddle, F. H. (1991). A regulatory region from the mouse Hox-2.2 promoter directs gene expression into developing limbs. Development 112,807 -811.[Abstract]
Srivastava, D., Cserjesi, P. and Olson, E. N. (1995). A subclass of bHLH proteins required for cardiac morphogenesis. Science 270,1995 -1999.[Abstract]
St Amand, T. R., Ra, J., Zhang, Y., Hu, Y., Baber, S., Qiu, M. S. and Chen, Y. P. (1998). Cloning and expression pattern of chicken Pitx2, a new component in the SHH signaling pathway controlling embryonic heart looping. Biochem. Biophys. Res. Commun. 247,100 -105.[CrossRef][Medline]
St Amand, T. R., Zhang, Y., Semina, E. V., Zhao, X., Hu, Y., Nguyen, L., Murray, J. C. and Chen, Y. P. (2000). Antagonistic signals between BMP4 and FGF8 define the expression of Pitx1 and Pitx2 in mouse tooth-forming anlage. Dev. Biol. 217,323 -332.[CrossRef][Medline]
Suzuki, M., Mizutani-Koseki, Y., Fujimura, Y., Miyagishima, H., Kaneko, T., Takada, Y., Akasaka, T., Tanzawa, H., Takihara, Y., Nakano, M. et al. (2002). Involvement of the Polycomb-group gene Ring1B in the specification of the anterior-posterior axis in mice. Development 129,4171 -4183.[Medline]
Takihara, Y., Tomotsune, D., Shirai, M., Katoh-Fukui, Y.,
Nishii, K., Motaleb, M. A., Nomura, M., Tsuchiya, R., Fujita, Y., Shibata, Y.
et al. (1997). Targeted disruption of the mouse homolog of
the Drosophila polyhomeotic gene leads to altered anteroposterior patterning
and neural crest defects. Development
124,3673
-3682.
Tie, F., Prasad-Sinha, J., Birve, A., Rasmuson-Lestander, A. and
Harte, P. J. (2003). A 1-megadalton ESC/E(Z) complex from
Drosophila that contains polycomblike and RPD3. Mol. Cell
Biol. 23,3352
-3362.
Ting-Berreth, S. A. and Chuong, C. M. (1996). Sonic Hedgehog in feather morphogenesis, induction of mesenchymal condensation and association with cell death. Dev. Dyn. 207,157 -170.[CrossRef][Medline]
Trevino, C., Anderson, R., Landry, M., Konig, G., Tonthat, B., Shi, C. and Muneoka, K. (1993). MPLB-2, a posterior signaling cell line derived from the mouse limb bud. Prog. Clin. Biol. Res. 383,295 -304.
Tsukui, T., Capdevila, J., Tamura, K., Ruiz-Lozano, P.,
Rodriguez-Esteban, C., Yonei-Tamura, S., Magallon, J., Chandraratna, R. A.,
Chien, K., Blumberg, B. et al. (1999). Multiple left-right
asymmetry defects in Shh (-/-) mutant mice unveil a convergence of
the Shh and retinoic acid pathways in the control of Lefty-1. Proc.
Natl. Acad. Sci. USA 96,11376
-11381.
Uchikawa, M., Ishida, Y., Takemoto, T., Kamachi, Y. and Kondoh, H. (2003). Functional analysis of chicken Sox2 enhancers highlights an array of diverse regulatory elements that are conserved in mammals. Dev. Cell 4,509 -519.[Medline]
Yamashita, H., Dijke, P., Huylebroeck, D., Sampath, T., Andries, M., Smith, J., Heldin, C. and Miyazono, K. (1995). Osteogenic protein-1 binds to activin type II receptors and induces certain activin-like effects. J. Cell Biol. 130,217 -226.[Abstract]
Yasuda, K., Momose, T. and Takahashi, Y. (2000). Application of microelectroporation for studies of chick embryogenesis. Dev. Growth Differ. 42,203 -206.[CrossRef][Medline]
Yochum, G. and Ayer, D. (2001). Pf1, a novel
PHD zinc finger protein that links the TLE corepressor to the mSin3A-histone
deacetylase complex. Mol. Cell. Biol.
21,4110
-4118.
Yokouchi, Y., Vogan, K. J., Pearse, R. V., II and Tabin, C. J. (1999). Antagonistic signaling by Caronte, a novel Cerberus-related gene, establishes left-right asymmetric gene expression. Cell 98,573 -583.[Medline]
Yoshioka, H., Meno, C., Koshiba, K., Sugihara, M., Itoh, H., Ishimaru, Y., Inoue, T., Ohuchi, H., Semina, E. V., Murray, J. C. et al. (1998). Pitx2. a bicoid-type homeobox gene, is involved in a lefty-signaling pathway in determination of left-right asymmetry. Cell 94,299 -305.[Medline]
Yoshitake, Y., Howard, T., Christian, J. L. and Hollenberg, S. M. (1999). Misexpression of Polycomb-group proteins in Xenopus alters anterior neural development and represses neural target genes. Dev. Biol. 215,375 -387.[CrossRef][Medline]
Yu, X. Y., St Amand, T. R., Wang, S. S., Li, G., Zhang, Y. D.,
Hu, Y. P., Nguyen, L., Qiu, M. S. and Chen, Y. P. (2001).
Differential expression and functional analysis of Pitx2 isoforms in
regulation of heart looping in the chick. Development
128,1005
-1013.
Zhang, Y. and Reinberg, D. (2001).
Transcription regulation by histone methylation, interplay between different
covalent modifications of the core histone tails. Genes
Dev. 15,2343
-2360.
Zhang, Z. Y., Yu, X., Zhang, Y. D., Geronimo, B., Lovlie, A., Fromm, S. H. and Chen, Y. P. (2000). Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation and duplication and posterior transformation of digit in mouse limb. Dev. Biol. 220,154 -167.[CrossRef][Medline]
Zhu, L., Marvin, M. J., Gardiner, A., Lassar, A., Mercola, M., Stern, C. D. and Levin, M. (1999). Cerberus regulates left-right asymmetry of the embryonic head and heart. Curr. Biol. 9,931 -938.[CrossRef][Medline]
|