Previous Article | Next Article 
Molecular and Cellular Biology, June 1999, p. 3958-3968, Vol. 19, No. 6
0270-7306/99/$04.00+0
Transcriptional Repression by XPc1, a New Polycomb
Homolog in Xenopus laevis Embryos, Is Independent of
Histone Deacetylase
John
Strouboulis,
Sashko
Damjanovski,
Danielle
Vermaak,
Funda
Meric, and
Alan P.
Wolffe*
Laboratory of Molecular Embryology, National
Institute of Child Health and Human Development, National
Institutes of Health, Bethesda, Maryland 20892-5431
Received 21 December 1998/Returned for modification 19 February
1999/Accepted 3 March 1999
 |
ABSTRACT |
The Polycomb group (Pc-G) genes encode proteins that assemble into
complexes implicated in the epigenetic maintenance of heritable patterns of expression of developmental genes, a function largely conserved from Drosophila to mammals and plants. The Pc-G
is thought to act at the chromatin level to silence expression of
target genes; however, little is known about the molecular basis of
this repression. In keeping with the evidence that Pc-G homologs in higher vertebrates exist in related pairs, we report here the isolation
of XPc1, a second Polycomb homolog in Xenopus laevis. We
show that XPc1 message is maternally deposited in a translationally masked form in Xenopus oocytes, with XPc1 protein first
appearing in embryonic nuclei shortly after the blastula stage. XPc1
acts as a transcriptional repressor in vivo when tethered to a promoter in Xenopus embryos. We find that XPc1-mediated repression
can be only partially alleviated by an increase in transcription factor dosage and that inhibition of deacetylase activity by trichostatin A
treatment has no effect on XPc1 repression, suggesting that histone
deacetylation does not form the basis for Pc-G-mediated repression in
our assay.
| |
INTRODUCTION |
The diversity required in higher
organisms to give rise to the multitude of differentiated cell types is
generated early in embryonic development. In Drosophila, the
best-studied example, the maintenance of homeotic patterns of
expression is dependent on the interplay of two antagonistic groups of
proteins, the Polycomb group (Pc-G) and the Trithorax group (Trx-G)
(49), with both groups comprising a large number of
genetically identified loci (31, 33). The role of the Pc-G
in the regulation of homeotic gene expression is one of repression:
Pc-G mutants exhibit posteriorly directed homeotic transformations
arising from the anteriorly ectopic expression of homeotic genes
(47, 70, 85). Conversely, the Trithorax group acts to
maintain transcriptional activity of homeotic genes (78,
79).
The developmental functions of the Pc-G appear to be conserved
throughout evolution, as evidenced by the isolation of Pc-G homologs in
mammals (67), Xenopus laevis (61),
chicken (87), plants (24, 25), and, recently,
Caenorhabditis elegans (30, 35). In addition, the
Polycomb phenotype can be rescued by expression of the murine M33
Polycomb homolog in mutant flies, providing further evidence for
functional conservation (45). The genetic analysis of Pc-G
function in null knockout mice has been particularly informative in
that respect. The range of phenotypes observed in Pc-G knockout mice
included posterior transformations in the axial skeleton, as well as
neurological and hematopoietic defects, all consistent with a role for
Pc-G in mammalian Hox gene regulation (67). These
diverse phenotypes provide strong evidence for extensive developmental
functions of Pc-G homologs in mice, at least some of which are
conserved between mammals and Drosophila.
Despite our increasing knowledge regarding the developmental functions
of the Pc-G, little is known about the underlying biochemical mechanisms. There is evidence suggesting that Pc-G members exist in
large nuclear multimeric protein complexes in Drosophila
(8, 20, 60) as well as in mammalian cells (1, 2, 26,
28, 63, 64, 66, 69, 82). There also appears to be considerable heterogeneity in Pc-G complex composition, distribution, and, potentially, function (8, 28, 69, 77, 82). Largely on the
basis of genetic evidence, parallels have been drawn between Pc-G-mediated repression and position effect variegation (PEV), a
heterochromatin-mediated repressive phenomenon in Drosophila (43, 56). This association between the Pc-G and PEV was
further strengthened at the molecular level by the identification of
the chromodomain, an N-terminal conserved protein motif originally shown to be shared by Polycomb, a key Pc-G member, and heterochromatin protein 1 (HP1), a component of constitutive heterochromatin and a
modifier of PEV (50). The chromodomain has been
subsequently shown to be present in an expanding class of heterogeneous
proteins, which appear to have chromosomal functions in common,
although not always related to transcriptional repression (10,
34).
Based primarily on the parallels drawn between Polycomb and HP1, it has
been suggested that the Pc-G acts at the chromatin level by forming a
multimeric repressive protein complex that spreads over entire domains,
reminiscent of heterochromatin in PEV (42, 43, 46, 47, 56).
Pc-G function might therefore provide an epigenetic means for
faithfully fixing and propagating the precise patterns of expression of
target genes, through multiple rounds of cell division in embryonic
development and tissue differentiation (10, 11, 55).
Precisely how this is achieved at the molecular level remains unknown.
Apart from the heterochromatin-like assembly model, one that is largely
superseded by recent evidence (6, 8, 74), a number of other
models have been proposed for Pc-G function. These include
compartmentalization of Pc-G target genes to nuclear subdomains of
transcriptional inactivity (8, 48); Pc-G-mediated
sequestration of gene regulatory elements, such as enhancers and
promoters, by looping out intervening DNA (6, 55, 56); or
localized remodelling of nucleosome density (54). The
localized modification of histones, such as deacetylation, was also
recently proposed to play a role in Pc-G mediated repression (10,
32a, 55).
The model system of X. laevis offers some advantages in the
study of the developmental regulation of chromatin components and their
impact on gene activity. For example, the developmentally regulated
release of masked maternal mRNA encoding histone H1 (14, 76,
86a) leads to the progressive accumulation of the somatic linker
histone through gastrulation. This causes the selective repression of
oocyte-type 5S rRNA genes (7, 32) and restrictions in the
capacity of ectodermal cells to change their fate to mesoderm (73,
83). As a step in employing Xenopus in the study of
Pc-G function, we report here the isolation of XPc1, a second Polycomb homolog in X. laevis. We find XPc1 to be distinct from the
previously described XPc homolog (61) and closely related to
the mouse M33 homolog. XPc1 gene expression is developmentally
regulated, and tethered XPc1 acts as a transcriptional repressor in
vivo in Xenopus embryos. Significantly, we found that
deacetylase inhibition by trichostatin A (TSA) treatment did
not have any effect on XPc1 repression in Xenopus embryos.
| |
MATERIALS AND METHODS |
Isolation of XPc1 cDNA.
A fragment corresponding to an
X. laevis Polycomb homolog was initially generated by
reverse transcription-PCR from ovary poly(A)+ RNA.
Degenerate primers accounting for codon bias in X. laevis were designed by using the cDNA for the mouse M33 Pc homolog as a
template (53). The 5' primer (5'-GCC/T GCC/T GAG/A TGC/T
ATC/T CTG AGC AAG-3') corresponds to M33 nucleotides 37 to 60, just inside the sequence encoding the conserved N-terminal chromodomain. The
3' primer (5'-GAT C/GAG GTT A/GGC TGT C/GAC ATG T/CGT-3') corresponds
to M33 nucleotides 1477 to 1500 derived from the sequence encoding the
C-terminal domain of homology (53). For both primers, the
degenerate nucleotides are separated from the M33 sequence by a slash.
From the M33 cDNA sequence, the predicted amplifiable fragment obtained
by using these primers is 1,463 nucleotides, covering the majority of
the coding sequence. Adult ovary poly(A)+ RNA was isolated
by using the Promega RNAgents kit according to the manufacturer's
instructions. Approximately 1 µg of oligo(dT)-primed poly(A)+ RNA was used for first-strand synthesis with avian
myeloblastosis virus reverse transcriptase according to the
instructions of the manufacturer (Promega, Madison Wis.). Approximately
one-fifth of the ovary cDNA was used as a template in each PCR with the degenerate primers at the stringent annealing temperature of 60°C. The predominant fragment amplified under these conditions was in the
predicted ~1.5-kb size range. This fragment was gel purified and
cloned into vector pGEM-T (Promega), and its identity was confirmed by
partial DNA sequencing of both ends. A PstI fragment containing the chromodomain was then used to screen a
Xenopus stage VI oocyte cDNA library constructed in Uni-ZAP
XR (Stratagene, La Jolla, Calif.). An excess of 1.25 × 106 phage plaques were transferred and immobilized on
Hybond-N+ filters (Amersham, Amersham, United Kingdom) and
were hybridized overnight at 65°C in 3× SSC (1× SSC is 0.15 M NaCl
plus 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS)-10×
Denhardt's solution-10% dextran sulfate-50 µg of sonicated salmon
sperm competitor DNA per ml. The filters were then washed several times
at 65°C in 0.3× SSC-0.1% SDS for 30 min each. A number of
candidate positive clones were isolated following successive rounds of
screening and were partially sequenced and arranged into overlapping
cDNAs. Two of these clones were used to assemble the full 4.37-kb XPc1 cDNA. Both strands of the full cDNA were completely sequenced by the
DNA Sequencing Core Facility at the Interdisciplinary Center for
Biotechnology Research of the University of Florida (Gainesville).
Northern blot analysis.
Total RNAs from oocytes isolated
from collagenase-treated ovary (71), adult tissues, and
embryos from different developmental stages was prepared by using RNA
STAT-60 according to the instructions of the supplier (Tel-Test B,
Friendswood, Tex.). Expression of XPc1, H1°, and H1C mRNAs was
assayed by standard Northern blot hybridization techniques with
approximately 5 µg of total RNA for each sample (3a, 33a).
Ribonucleoprotein fractionation by density gradient
centrifugation.
Extract from 100 oocytes or embryos was
fractionated on a Nycodenz (Nycomed, Oslo, Norway) density gradient
(41). Fractions were stripped of proteins by
phenol-chloroform extraction, and RNA was recovered by ethanol
precipitation. RNA pellets were dissolved in a small volume of water,
and fractionation profiles of specific mRNAs were determined by
Northern blot hybridization as described above.
XPc1 antibody production and Western blot analysis.
Affinity-purified XPc1 antibodies were obtained from rabbits immunized
with the synthetic peptide RNPRPRDSHPVPQKKAPA, corresponding to amino
acids 125 to 142 of XPc1. Peptide synthesis, purification, and
coupling; rabbit immunizations; initial serum screening; and antibody
affinity purification were all carried out by Quality Control
Biochemicals, Inc. (Hopkinton, Mass.). For XPc1 protein detection by
immunoblotting, extracts were prepared (37), and proteins
were separated by SDS-polyacrylamide gel electrophoresis on an 8%
Tris-glycine-SDS gel and electroblotted onto Hybond-ECL nitrocellulose
filters (Amersham). Filters were routinely incubated at 4°C overnight
with a 100-fold dilution of XPc1 antibody. Detection was by
chemiluminescence with SuperSignal (Pierce, Rockford, Ill.).
Isolation of nuclei from Xenopus embryos.
Nuclei
were prepared (4). Briefly, 50 to 100 embryos from different
developmental stages were homogenized in 300 µl of E-1 buffer (110 mM
KCl, 50 mM Tris-HCl [pH 7.4], 5 mM MgCl2, 0.1 mM
spermine, 0.1 mM EDTA, 2 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl fluoride)-0.25 M sucrose, and the final volume was adjusted to 500 µl with the same buffer. To this homogenate, 2.4 ml of E-1-2.2 M
sucrose buffer was added, mixed, and then layered onto a 150-µl cushion of E-1-2.2 M sucrose buffer in a polyallomer centrifuge tube
(13 by 51 mm; Beckman, Fullerton, Calif.). Nuclei were pelleted by
centrifugation at 130,000 × g and 4°C for 2 h
with a TLA100.3 rotor in a Sorvall TLA benchtop ultracentrifuge.
Pelleted nuclei were resuspended in 250 µl of nuclear buffer (25%
glycerol, 25 mM Tris-HCl [pH 7.4], 70 mM KCl, 5 mM MgCl2,
0.2 mM EDTA, 2 mM dithiothreitol, 0.4 mM phenylmethylsulfonyl
fluoride), and residual yolk protein was removed by washing the nuclei
twice by pelleting through a 10-µl cushion of 80% glycerol at 6,000 rpm in a Microfuge at 4°C for 10 min. In order to remove most of the
melanin granules that copellet with the nuclei during the
ultracentrifugation step, nuclear pellets were resuspended in 500 µl
of ice-cold phosphate-buffered saline and repelleted at 1,500 rpm for 5 min in a benchtop centrifuge at 4°C. Nuclei were then resuspended in
50 to 100 µl of nuclear buffer and lysed by sonication.
In vitro transcription.
All in vitro-transcribed
polyadenylated RNAs were generated by using the SP6 transcription
vector pSP64 (Promega), which was modified by the insertion of a linker
in the unique EcoRI site downstream of the poly(A) stretch.
This linker provides additional restriction sites for linearizing the
vector prior to transcription (the modified vector was kindly donated
by Melissa Stolow). Capped RNA transcripts were generated by using the
SP6 mMessage mMachine kit according to the instructions of the
manufacturer (Ambion, Austin, Tex.). For in vitro transcription, the
XPc1 cDNA was cloned into the HindIII/HincII
sites of the modified pSP64. N-terminally tagged XPc1 was constructed
by amplifying three copies of the HindIII-fitted
hemagglutinin (HA) epitope tag from plasmid pSM491 (81) and
cloning it into the HindIII site of XPc1pSP64. The GAL4
DNA binding domain (DBD) was cloned into the HindIII
site of XPc1pSP64 by Deep Vent DNA polymerase (New England Biolabs, Beverly, Mass.) amplification with specific primers fitted with HindIII restriction sites. This results in an in-frame
fusion of the GAL4 DBD to the N terminus of XPc1. In addition, the GAL4 DBD alone was cloned into the HindIII/PstI
sites of the modified pSP64 vector by PCR with Deep Vent DNA polymerase
and primers fitted with the appropriate restriction sites. The in vitro
transcription construct for Xenopus heat shock factor (HSF)
(75) has been previously described (38). The
FLAG-tagged histone H1 construct has been previously described
(7). Constructs were checked for correct in-frame fusions by
DNA sequencing. In addition, in vitro-transcribed RNAs were
microinjected into oocytes and checked by Western blotting with
anti-XPc1, anti-GAL4 (Babco, Richmond, Calif.), anti-HA (Boehringer
Mannheim, Indianapolis, Ind.), and anti-FLAG M2 (Kodak IBI, Rochester,
N.Y.) antibodies.
XPc1 phosphorylation and nuclear localization.
In
vitro-transcribed RNA for full-length XPc1 was injected into oocytes as
described below. Following incubation to allow for protein synthesis,
germinal vesicles (GVs) were manually dissected and the remaining
oocyte cytoplasms were collected. Alkaline phosphatase assays were
carried out as described previously (52). Briefly, protein
extracts were precipitated with 10% trichloroacetic acid, and pellets
were washed twice with cold acetone. Pellets were then resuspended in
30 mM triethanolamine (pH 8.25)-0.1 mM EDTA-1 mM
MgCl2-0.2% SDS buffer and divided into two aliquots. To
one aliquot 10 U of alkaline phosphatase (Boehringer Mannheim) was added, followed by incubation at 37°C for 2 h. The second
aliquot was mock treated by incubation in the same way but with no
phosphatase addition.
Embryo and oocyte microinjections.
For embryo
microinjections, mature Xenopus females were injected with
1,000 IU of human chorionic gonadotropin (Sigma, St. Louis, Mo.). After
12 h at 18 to 22°C, the females were stripped of eggs which were
fertilized in vitro (39). Fertilized eggs were dejellied in
2% cysteine (pH 8.0), followed by several washes in MMR buffer
(62). Washed fertilized eggs were placed in 1× MMR-5%
Ficoll (Pharmacia, Piscataway, N.J.) and allowed to begin their first
cleavage division before they were microinjected. In all experiments,
500 to 800 pg of DNA was injected in a total volume of 13.8 nl per
embryo, either alone or with in vitro-synthesized RNA. In all cases, a
maximum of approximately 1 ng of each in vitro-transcribed RNA was
coinjected with the DNA. Injected embryos were placed in 0.1× MMR-2%
Ficoll and incubated at 25°C until harvested at various developmental
stages for analysis. For TSA treatments, embryos were placed in medium
with 30 or 90 nM TSA immediately after microinjection. TSA was
purchased from Wako Pure Chemical Industries (Tokyo, Japan) and
dissolved in ethanol at a stock concentration of 30 µM. The H10
chloramphenicol acetyltransferase (CAT), cytomegalovirus CAT, hsp70CAT,
and G5hsp70CAT reporter plasmids have been previously described
(3a, 33a, 52, 59). Oocyte microinjections were as previously
described (3a). Microinjection of RPD3 mRNA into oocytes and
embryos was exactly as described earlier (7, 86a).
RNA analysis by primer extension.
For the XPc1 tethering
repression assays, healthy injected embryos were collected at various
times after fertilization and homogenized as described previously
(37). Primer extension analysis with RNA equivalent to three
embryos was carried out as previously described (80). Three
primers were utilized: first, we used a CAT-specific primer
(37) which gives rise to a 167-nucleotide extension product
from the hsp70 promoter; second, to assay H1° transcription, we used
a 26-mer (5'-GTCAAGCCCTGACTCGCAATGGCTTC-3') that hybridizes
in the noncoding region of the H1° gene; and finally, as an RNA
recovery and loading control, we used a histone H4 primer (5'-GAG GCC
GGA GAT GCG CTT GAC-3') which hybridizes to endogenous H4 RNA, giving
rise to a 182-nucleotide extended product. All microinjections and RNA
analyses were carried out at least in duplicate with reproducible
results (relative transcription levels varied less than 5%).
| |
RESULTS |
Isolation and sequence analysis of a second Xenopus
Polycomb homolog.
In order to isolate a Polycomb homolog in
Xenopus, we employed reverse transcription-PCR with a set of
degenerate primers that were designed by using the mouse M33 homolog as
a template (53). A cDNA fragment of the predicted size was
amplified from adult ovary mRNA and cloned. Partial sequencing
confirmed that the cloned cDNA fragment was a putative
Xenopus Polycomb homolog. This fragment was used as a probe
to screen a Xenopus oocyte cDNA library. This resulted in
the isolation of several overlapping clones that were used to assemble
a 4,380-bp cDNA clone (GenBank Accession number AF101438). This clone
has a single 1,416-bp open reading frame coding for a predicted
472-amino-acid protein. Interestingly, the majority (approximately 2.9 kb) of the cDNA is 3' untranslated sequence, suggesting that
posttranscriptional control of gene expression may occur.
Amino acid analysis revealed that the predicted protein is a Polycomb
homolog. First, it possesses an evolutionarily conserved N-terminal
chromodomain that is 60% identical (75.5% similar, with conservative
changes included) to the Drosophila Pc chromodomain and 93%
identical to the mouse M33 chromodomain (Fig. 1A, B, and C). The chromodomain of the protein
reported here clearly belongs to the Polycomb class, as evidenced, for
example, by the presence of the ILDPRLL amino acid identity which is
found immediately downstream of the chromodomain in Polycomb homologs
(Fig. 1A and B). In addition, Pearce et al. (53) identified
a short region of C-terminal homology (the COOH box) which is shared
between Polycomb homologs and which is also present in the protein
reported here (Fig. 1D). Finally, from an extensive analysis of
chromodomain-containing proteins, Koonin et al. (34)
proposed that proteins in the Polycomb class share a similar overall
structure, in which the conserved N- and C-terminal domains (the
chromodomain and COOH box, respectively) are contained within short
globular domains separated by a long nonglobular central region. The
predicted protein reported here fully conforms to the proposed
structure. We conclude, therefore, that the cDNA we isolated codes for
a true Xenopus Polycomb homolog that we will henceforth
refer to as XPc1 (for Xenopus Polycomb homolog 1) (see also
below).
View larger version (66K):
[in this window]
[in a new window]
|
FIG. 1.
(A and B) Amino acid sequence comparison of XPc1 to
Xenopus XPc2 (A) and mouse MPc1 (B) Polycomb homologs. The
conserved chromodomains and COOH boxes are shaded. Conserved putative
nuclear localization signals are underlined. The sequence of the
synthetic peptide used to raise antibodies against XPc1 is shown in
boldface in panel A; the polyserine stretch conserved between XPc1 and
M33 is in boldface in panel B. (C) Amino acid alignments of the
conserved chromodomains of higher vertebrate Polycomb homologs and
of Drosophila Pc. The shaded areas indicate identity with
the indicated consensus sequence. Lowercase letters indicate amino
acids that deviate from the consensus sequence. (D) Amino acid
alignments of the C-terminal COOH boxes of vertebrate Polycomb
homologs and Drosophila Pc. The shaded areas in the COOH-box
indicate identity with the consensus sequence, and lowercase letters
indicate residues that are not identical to the consensus. Additional
sequences beyond the conserved COOH box are boxed (XPc1, MPc1, and
hPc1) or underlined (XPc2, MPc2, hPc2) in order to illustrate the
extended C-terminal homologies which allow the classification of the
vertebrate Polycomb homologs into two classes. MPc1 is M33
(53), and hPc1 is CBX2 (23) (see text for
details). All sequence analyses were done by using the Wisconsin
Genetics Computer Group package.
|
|
The isolation of another Polycomb homolog in
Xenopus (XPc)
has been reported previously (
61). Sequence comparison of
our
XPc1 homolog with that isolated by Reijnen et al. (
61)
revealed
surprisingly little identity between the two proteins outside
the conserved N-terminal chromodomain and C-terminal COOH box
(Fig.
1A). Overall, the two proteins have only 28.5% identity,
or 43.8%
similarity when conservative amino acid changes are included.
Both
Xenopus homologs are similarly conserved with
Drosophila Pc (45% similarity for XPc versus 42%
similarity for XPc1). By
contrast, XPc1 is much more closely related to
the mouse M33 homolog
(
53), with homology extending well
beyond the conserved N- and
C-terminal motifs (Fig.
1B). It is
striking, for example, that
both XPc1 and M33 contain a conserved
polyserine stretch near
the N terminus of the protein, which is not
present in any other
Polycomb homolog (Fig.
1B). Overall, XPc1 and M33
have 53% identity,
or 67.5% similarity. XPc1 and M33 also exhibit
similar degrees
of homology to the other mammalian Polycomb homologs.
For example,
they are both closely related to the human homolog CBX2
(
23)
(recently renamed hPc1 [
64]). By
contrast, both M33 and XPc1
are more distantly related to the recently
described mammalian
homologs MPc2 (
2) and hPc2
(
64). The
Xenopus Polycomb homolog
described by
Reijnen et al. (
61) is instead more closely related
to MPc2
and hPc2 (
2,
64), and we would therefore like to
rename it
XPc2. XPc2, MPc2, and hPc2 share a characteristic short
amino acid
motif, YVTV, immediately following the COOH box, which
is not present
in XPc1, M33 (MPc1), or CBX2 (hPc1). They instead
have more extensive
regions of sequence identity extending further
upstream and downstream
of the COOH box. Although overall sequence
identity has already served
to divide vertebrate Polycomb homologs
into two classes
(
63), we suggest that the blocks of extended
C-terminal
homology could serve as a further, more specific, means
of
distinguishing between
them.
XPc1 gene expression: masked maternal mRNA and XPc1 accumulation in
embryonic nuclei.
We determined the temporal patterns of
expression and tissue distribution of the XPc1 transcript by Northern
blot analysis. With the XPc1-coding region as a probe, an approximately
4.5-kb transcript is detected in all samples assayed; this size is
consistent with that of the cloned XPc1 cDNA. The message for XPc1
appears to be abundant in the early stages of oocyte development and
remains abundant in unfertilized eggs and early in embryonic
development (data not shown). The abundance of XPc1 message appears to
decline around gastrulation, remaining low during neurulation and
moderately increasing again around the tailbud stage and in subsequent
developmental stages. The relative abundances of XPc1 mRNA in oocytes,
eggs, and embryonic stages prior to the midblastula transition (MBT) strongly suggests that the XPc1 mRNA in these stages is of maternal origin, as has also been suggested for XPc2 (61). The
overall pattern of XPc1 expression during Xenopus
development is similar to that observed for XPc2 (61). It is
estimated that as much as 80% of the maternal mRNA synthesized in
Xenopus oocytes is masked, complexed with storage messenger
ribonucleoproteins (mRNPs), only to be translationally mobilized in the
early stages of embryonic development (12). It should be
emphasized that the oocyte exhibits highly selective tissue-specific
patterns of gene expression. In general, mRNAs that are synthesized and
sequestered in masked form encode proteins whose functions help
determine differentiated states of gene expression in somatic cells
(73, 83). The fact that the XPc1 cDNA contains a large
(~2.9-kb) 3' untranslated region (3' UTR) and the presence of a long
U tract (37 bases) in the 3' UTR led us to examine whether XPc1 is a
maternal mRNA that is masked in the oocyte and translationally
activated late in development (72). We found that the XPc1
mRNA in Xenopus oocytes is exclusively associated with the
translationally inactive storage mRNPs (Fig.
2A, upper panel). It is
striking that the XPc1 message appears to be very tightly associated
with one particular fraction (fraction 11) (Fig. 2A, upper panel). This
may reflect an intimate association of XPc1 mRNA with a component(s) of
the mRNP complexes. By contrast, the mRNA for the translationally active transcription factor TFIIIA cofractionates with mRNP and ribosomal fractions (Fig. 2A, lower panel), as previously observed (76). The masking of XPc1 message in the mRNP complex is
maintained to the blastula stage (Fig. 2B). This is remarkably late in
development for a masked maternal mRNA and may reflect the distance of
the U tract from the 3' end of the mRNA (72). Our results
suggest that XPc1 protein will begin to accumulate in
Xenopus embryos only after the blastula stage is complete.
View larger version (36K):
[in this window]
[in a new window]
|
FIG. 2.
(A) XPc1 mRNA masking in Xenopus oocytes.
Total mRNA from stage VI Xenopus oocytes was fractionated on
a Nycodenz density gradient. Fractions were analyzed by Northern blot
hybridization. The blot in the top panel shows the XPc1 message to be
associated with translationally inactive storage mRNPs. As a control,
the same filter was stripped and reprobed with TFIIIA to show the
association of this message with ribosomes as well as with mRNPs
(bottom panel). (B) Same as panel A except that mRNPs were fractionated
from blastula-stage embryos (6 h postfertilization). (C) Western blot
analysis with anti-XPc1 antibody to show that XPc1 protein (arrow) is
first detected in em- bryonic nuclei after the blastula stage. Lanes 1 to 4, nuclear extracts from different developmental stages (10 h
postfertilization). Blast., blastula; Gastr., gastrula; Neur., neurula;
Stg., stage. Lane 5, total embryonic protein extract from embryos (10 h
postfertilization) microinjected with XPc1 in vitro-transcribed RNA.
Lane 6, noninjected (Non inj.) total embryo extract. M, markers.
|
|
In order to determine the timing of the appearance of XPc1 protein
during
Xenopus development and as a means of extending
the
observations on XPc1 mRNA masking, we raised a rabbit polyclonal
antibody against a synthetic peptide derived from the N terminus
of the
predicted amino acid sequence of XPc1 (see Materials and
Methods). This
peptide shows little homology with the XPc2 protein
(Fig.
1A). The
antibody against the synthetic peptide detects
a band of approximately
70 kDa, or more, in protein extracts from
embryos (8 to 9 h
postfertilization) injected with in vitro-transcribed
XPc1 RNA (Fig.
2C, lane 5). No band in this size range is detected
in noninjected
embryos (Fig.
2C, lane 6) or in oocytes (not shown).
This suggested
that XPc1, if at all present, is not very abundant
in embryos and
prompted us to test embryonic nuclei from different
developmental
stages. In addition, our antibody was raised against
a synthetic
peptide and recognizes several other proteins in whole
embryonic
extracts, so restricting our analysis to nuclear proteins
would enable
us to focus on XPc1. Endogenous XPc1 protein was
not detected in the
oocyte or in the GV (the oocyte's nucleus).
This is in good agreement
with our finding that XPc1 mRNA is stored
in a translationally inactive
form in the oocyte (Fig.
2A). In
addition, XPc1 protein did not appear
to be present in embryonic
nuclei at the MBT, the stage at which the
zygotic genome becomes
transcriptionally active, which is consistent
with the mRNP masking
data (Fig.
2B and Fig.
2C, lane 1). A protein
band of a size similar
to that detected in XPc1-injected embryos first
becomes detectable
in gastrula-stage nuclei (Fig.
2C, lane 2) and
persists up to
the early tailbud stage (Fig.
2C, lane 4), which is the
latest
developmental time point tested in this experiment. At
approximately
70 kDa, the XPc1 protein detected in embryonic nuclei, as
well
as in injected embryos, is appreciably larger than the predicted
52 kDa. This is consistent with the sizes observed for all Polycomb
homologs characterized to date (
2,
51,
64) and has been
attributed to a high content of charged amino acids, posttranslational
modifications, or both (
51). We next wished to examine
whether
the regulation of XPc1 uptake into nuclei reflected a
deficiency
in nuclear import of XPc1 in oocytes and early embryos or
whether
oocytes were competent to accumulate XPc1 in nuclei. We also
wished
to examine whether the aberrant mobility of XPc1 (Fig.
2C)
reflects
posttranslational
modification.
XPc1 synthesized from exogenous mRNA microinjected into oocyte
cytoplasm readily accumulates in the GVs of injected oocytes
(data not
shown). This result indicates that the XPc1 protein
is competent for
nuclear import. It should be noted that masking
of maternal mRNA
synthesized in vivo depends on both transcription
and association with
the FRGY2 protein (
7). Thus, naked mRNA
injected into
oocytes is translated. The XPc1 protein that accumulates
in oocyte
nuclei and cytoplasm migrates aberrantly with an apparent
size of at
least 70 kDa, as seen in embryos (Fig.
2C and data
not shown). Alkaline
phosphatase treatment of nuclear and cytoplasmic
XPc1 results in an
increase in electrophoretic mobility (data
not shown). This result
indicates that the XPc1 is a phosphoprotein.
Thus, the aberrant
mobility of XPc1 can be partially explained
by phosphorylation, and the
protein synthesized in oocytes is
competent for nuclear uptake. We next
focused our attention on
the mechanism of XPc1-mediated transcriptional
repression.
XPc1 is a transcriptional repressor in Xenopus
embryos.
Polycomb has not been shown to bind to DNA directly
(42). However, immunostaining analysis in
Drosophila salivary glands has shown that it associates with
several (over 100) loci in polytene chromosomes (88). We
tested whether injected HA epitope-tagged XPc1 could be detected in
embryonic chromatin. As a control for chromatin localization, we
coinjected FLAG-tagged histone H1 in vitro-transcribed RNA. Embryos
were collected at the early gastrula stage and treated with limiting
amounts of micrococcal nuclease. Embryonic chromatin of different
nucleosomal lengths was fractionated by sucrose gradient centrifugation
(22), and individual fractions were assayed for the lengths
of nucleosomal fragments (data not shown). By assaying for the
fractionation profiles of HA-XPc1 and H1-FLAG, we found that XPc1
fractionated almost exclusively with the soluble, nucleosome-free
fractions, whereas H1-FLAG could clearly be detected in the
polynucleosomal chromatin fractions (data not shown). From this we
conclude that injected XPc1 cannot be detected in the bulk chromatin of
Xenopus embryos.
Polycomb has been genetically characterized as acting as a
transcriptional repressor, yet it does not bind to DNA directly.
When
fused to a DBD and artificially tethered to a promoter, however,
Polycomb and its higher vertebrate homologs have been shown to
act as
repressors in transiently (
9,
63,
64,
66) or stably
(
2) transfected cells and in transgenic flies
(
45). We wished
to determine whether XPc1 would also act as
a transcriptional
repressor in
Xenopus embryonic chromatin
when tethered to a promoter.
We fused the yeast GAL4 DBD (amino acid
residues 1 to 147) to
the N terminus of XPc1 so as not to interfere
with transcriptional
repression, which appears to be mediated by the
C-terminal domain
of the protein (
9,
44,
64,
66). The
reporter we chose
to use for our transcriptional assays was that of the
Xenopus hsp70 promoter (
5). The transcriptional
regulation and
cis-acting
requirements of this promoter have
been characterized in a chromatin
context for
Xenopus
oocytes, embryos, and somatic cells (
37-40).
Furthermore,
the hsp70 promoter can be induced to high levels
of transcription in
all of these systems by a simple heat shock
treatment (
37).
In the absence of heat shock, the hsp70 promoter
is capable of high
levels of transcription in oocytes by exogenous
addition of
Xenopus HSF (
38,
75). We used a reporter plasmid
in which five copies of the GAL4 DNA binding sites were cloned
in
tandem immediately upstream of the hsp70 promoter, which was
fused to a
CAT reporter gene
(G5hsp70CAT).
We employed
Xenopus embryos to examine the influence of
GAL4XPc1 on transcription. We first tested the localization of GAL4XPc1
in the nuclei of microinjected embryos. In Fig.
3A (lane 1), it
is
evident that microinjected GAL4XPc1 is efficiently localized
in
embryonic nuclei, even at developmental time points when endogenous
XPc1 is not detectable. We next tested the transcriptional regulation
of the G5hsp70CAT reporter plasmid in embryos by coinjecting the
DNA
with in vitro-transcribed HSF mRNA (Fig.
3B). No transcription
from the
hsp70 promoter can be detected at the MBT (Fig.
3B, lane
1), possibly
due to the transcriptional constraints to which the
zygotic genome is
subjected up to that stage of development (
3,
39,
57,
58).
However, robust levels of hsp70 transcription
appear shortly after the
blastula stage (Fig.
3B, lane 2) and
persist up to the gastrula stage
(Fig.
3B, lane 5). Tethering
GAL4XPc1 to the hsp70 promoter almost
completely abolishes transcription
at all developmental time points
tested (Fig.
3B, lanes 6 to 10)
even in the continued presence of HSF.
Coinjection of XPc1 RNA
without the GAL4 DBD tether leads to only a
slight reduction in
hsp70 transcriptional levels (Fig.
3B, lanes 11 to
15). This is
probably a nonspecific effect due to the sensitivity of
Xenopus embryos to the dosage of microinjected nucleic acids
(
62). We
conclude from this experiment that tethering
GAL4XPc1 to the hsp70
promoter efficiently represses transcription
following zygotic
gene activation at the MBT (
27). The
results after removal of
the GAL4 DBD tether suggest that this
repression is specific and
not due to the indiscriminate overexpression
of proteins in microinjected
embryos. The repressive effect of GAL4XPc1
is not peculiar to
the hsp70 promoter, as we observed a similar
repression in embryos
when GAL4XPc1 was tethered to a herpes simplex
virus thymidine
kinase promoter construct (data not shown).
View larger version (40K):
[in this window]
[in a new window]
|
FIG. 3.
Transcriptional repression by tethering of GAL4XPc1 to
an hsp70CAT reporter construct in Xenopus embryos. (A) The
GAL4XPc1 fusion protein is efficiently localized in embryonic nuclei.
Lanes 1 to 4, nuclear extracts at developmental stages between the MBT
and gastrula (Gastr.) from embryos microinjected with GAL4XPc1 RNA.
Lane numbers indicate hours postfertilization. The XPc1 antibody used
detects endogenous XPc1 as well as the GAL4XPc1 fusion protein
(arrows). Lane 5, total protein extract (E.) from embryos microinjected
with GAL4XPc1. Lane 6, noninjected (N.I.) total extract control. Lane
7, noninjected nuclear extract from neurula embryos, used as a positive
control for detecting endogenous XPc1. M, markers. (B) Primer extension
analysis of transcription from the microinjected hsp70CAT reporter
during early Xenopus development is shown in lanes 1 to 5. Microinjected embryos were collected and analyzed at hourly time points
between the MBT (approximately 7 h postfertilization) and the
gastrula stage (approximately 11 h postfertilization). In order to
drive detectable levels of hsp70 transcription, in vitro-transcribed
Xenopus HSF RNA was coinjected with the reporter plasmid in
all experiments. The effect of tethering of GAL4XPc1 to the hsp70
promoter is shown in lanes 6 to 10. Lanes 11 to 15 show the absence of
significant repression from the hsp70 promoter when XPc1 is coinjected
without the tether of the GAL4 DBD. A.U., arbitrary units. (C) Effects of
titrating GAL4XPc1 (lanes 1 to 3) versus the GAL4 DBD (lanes 4 to 6) on
hsp70 transcription in microinjected embryos. Approximately equal
amounts of in vitro-transcribed GAL4XPc1 or GAL4 DBD RNA were injected.
Due to the size difference between the two RNAs and assuming similar
translation rates, the GAL4 DBD is in approximately a fourfold molar
excess compared to GAL4XPc1 in these experiments. Lane 7, hsp70CAT-HSF
coinjection control.
|
|
To assess whether the repressive effect on the hsp70 promoter was due
to a nonspecific squelching effect mediated by the GAL4
DBD fused to
XPc1, we injected increasing, but equivalent, amounts
of GAL4XPc1 or
GAL4 DBD in vitro-transcribed RNAs (Fig.
3C). Transcriptional
activity
was assayed in embryos at 10 h postfertilization. It
can be seen
that whereas GAL4XPc1 almost completely repressed
hsp70 transcription
at all RNA concentrations injected, the GAL4
DBD had only a very
moderate effect on transcription at the highest
concentration (Fig.
3C). It should be noted that due to the size
difference for the two
RNAs, the GAL4 DBD in these injections
is in a fourfold molar excess
compared to GAL4XPc1. We reason
from this that the GAL4 DBD does not
significantly contribute
to the observed repression by GAL4XPc1. In
addition, our GAL4XPc1
titration indicates that relatively low levels
of mRNA can serve
to repress transcription, and our Western blotting
analysis indicates
that XPc1 levels are raised less than 10-fold over
those present
in control embryos. From the evidence presented in Fig.
3B and
C, we conclude that the GAL4XPc1-mediated repression of the
hsp70
promoter in
Xenopus embryos is specific to the XPc1
part of the
fusion
protein.
It has been previously shown that heat shock at 34°C induces high
levels of transcription from an hsp70 promoter injected
in embryos
(
36,
38). In general, heat shock mobilizes endogenous
HSF
residing in the cell in an inactive complexed form. We wanted
to test
whether the heat shock-inducible increase in the dosage
of HSF would be
sufficient to alleviate the repressive effect
of GAL4XPc1 on the hsp70
promoter. As a control for heat shock,
we injected G5hsp70CAT plasmid
DNA alone (Fig.
4A, lanes 1 to
3). As
previously seen (
37), there is little hsp70 transcription
in
the absence of exogenously added HSF (Fig.
4A, lane 1). Heat
shock for
as little as 30 min, however, is sufficient to induce
high levels of
hsp70 transcription (lane 2), which increases further
with prolonged
heat shock treatment (lane 3). Coinjection of HSF
mRNA together with
the G5hsp70CAT reporter further enhances the
heat shock response (Fig.
4A, lanes 5 and 6). By contrast, tethering
GAL4XPc1 reduces the heat
shock responsiveness of the hsp70 promoter
such that only a moderate
increase in transcription is observed
(Fig.
4A, lanes 8 and 9), which,
even after prolonged heat shock
treatment, does not reach control
non-heat-shock-induced levels
(compare lane 4 to lane 9). Increasing
the dosage of the HSF transcription
factor, therefore, does not fully
reverse the repressive effect
of GAL4XPc1 on the hsp70 promoter in
Xenopus embryos.
View larger version (59K):
[in this window]
[in a new window]
|
FIG. 4.
GAL4XPc1-mediated repression of transcription is
maintained in the presence of elevated levels of HSF and in the
presence of inhibitors of histone deacetylase. (A) Effect of heat shock
(HS) on GAL4XPc1-mediated repression in microinjected embryos. Lanes 1 to 3, inducibility of the hsp70 promoter in the absence of any
coinjected HSF after 30 (lane 2) or 60 (lane 3) min of heat shock.
Lanes 4 to 6, enhanced heat shock induction of the hsp70 promoter when
coinjected with HSF RNA. Lanes 7 to 9, heat shock is not sufficient to
restore hsp70 transcription to control levels when GAL4XPc1 is tethered
to the promoter. A.U., arbitrary units. (B) Effect of TSA treatment on
GAL4XPc1-mediated repression. Microinjected embryos were treated with
30 nM (lanes 2, 5, 8, and 11) or 90 nM (lanes 3, 6, 9, and 12) TSA and
harvested for analysis before (stage 10, lanes 1 to 3 and 7 to 9) or
after (stage 13, lanes 4 to 6 and 10 to 12) gastrulation (gastr.). For
all experiments, unless otherwise indicated, embryos were harvested for
analysis at approximately the initial gastrula stage (stage 10). A
histone H4 primer was included in all primer extensions as a control
for RNA recovery and loading. (C) Expression of RPD3 represses
transcription from the H1° promoter, and TSA relieves this
repression. Oocytes were injected with double-stranded DNA of H1°
with or without an increasing amount of RPD3 mRNA (0.5 ng in lanes 2 and 6 and 1 ng in lanes 3 and 7). The oocytes were assayed by primer
extension. The oocytes were incubated overnight in the presence of 30 nM (+) or 90 nM (++) TSA or in the absence of TSA ( ). The positions
of the extension products of the H1° transcript and of the endogenous
H4 control are indicated. (D) Lanes 1 and 2, fertilized eggs were
microinjected with 1 ng of RPD3 mRNA (+) or water () and allowed to
develop. Lanes 3 and 4, fertilized eggs were incubated in the absence
() or presence (+) of 30 nM TSA. Total RNA was isolated from 10 embryos and electrophoresed on a 1% agarose gel. The blot was probed
with [ -32P]dCTP random-prime-labeled full-length H1°
or H1C coding regions and washed stringently.
|
|
We next tested whether inhibition of the RPD3 family of histone
deacetylases (
33b) by TSA was sufficient to reverse
XPc1-mediated
repression. Three lines of evidence led us to carry out
this experiment.
First, recent work in our laboratory showed that
treatment with
low levels of TSA could induce high levels of hsp70
transcription
in
Xenopus oocytes, comparable to those
achieved by heat shock
(
40). Second, it was recently shown
that the transcriptional
repression observed in
Schizosaccharomyces pombe centromeric heterochromatin
domains could be alleviated by treatment with TSA (
19). The
repressive effect in
S. pombe heterochromatin is mediated by
swi6,
a chromodomain-containing homolog of HP1 (
18). Third,
the
Drosophila dMi-2 protein is a Hunchback-interacting
protein that functions
in Polycomb-mediated repression
(
32a), and
Xenopus Mi-2 is part
of a histone
deacetylase complex (
83b). Bearing in mind the parallels
that have often been drawn between heterochromatic and Pc-G repression,
it was suggested that deacetylation may also play a role in
Pc-G-mediated
repression (
10,
33a). We therefore tested
whether TSA treatment
was sufficient to lift the GAL4XPc1-mediated
repression on the
hsp70 promoter in
Xenopus embryos.
Immediately following microinjection,
embryos were treated with two
concentrations of TSA, 30 and 90
nM. Deacetylase activity is
effectively inhibited at both TSA
concentrations in embryos
(
83a) (see Fig.
4C and D below). Surprisingly,
both
concentrations of TSA had little effect on hsp70 transcription
prior to
gastrulation (Fig.
4B, lanes 1 to 3, 8 h postfertilization).
After
gastrulation (14 h postfertilization), we observed an induction
of
hsp70 transcription, the levels of which were proportional
to the TSA
concentration used (Fig.
4B, lanes 4 to 6). By contrast,
TSA treatment
appears to have practically no effect on GAL4XPc1-mediated
repression,
before or after gastrulation (Fig.
4B, lanes 7 to
12). In order to
control for the effectiveness of TSA in inhibiting
the RPD3 class of
histone deacetylases inhibitors during early
embryogenesis, we made use
of the H1° promoter (
3a,
33a). We
tested the role of
Xenopus RPD3 in H1° promoter regulation by
examining
transcription in oocytes microinjected with increasing
amounts of RPD3
mRNA (Fig.
4C, lanes 1 to 3). Increasing amounts
of RPD3 mRNA elevate
deacetylase activity in oocytes and repress
transcription from the
H1° promoter. This repression is relieved
in the presence of TSA
(Fig.
4C, lanes 4 to 7). Similar results
are obtained with embryos when
microinjection of RPD3 mRNA selectively
represses H1° mRNA
transcription from the endogenous chromosomes
(Fig.
4D, lanes 1 and 2),
whereas addition of TSA to embryos as
for Fig.
4B selectively activates
H1° transcription (Fig.
4D,
lanes 3 and 4) (
3a). We
conclude that deacetylation is unlikely
to play a significant role in
Polycomb-mediated transcriptional
repression in our in vivo assay with
Xenopus embryos.
| |
DISCUSSION |
We report here the isolation and characterization of XPc1, a new
chromodomain homolog in X. laevis. XPc1 is the second
Polycomb homolog to be isolated in Xenopus, in agreement
with the accumulating evidence that Pc-G vertebrate homologs exist in
pairs (2, 63, 64). It is also clear that Pc-G homolog pairs,
although related, are distinct proteins (2, 26, 61, 63, 64).
On the basis of sequence homology, the vertebrate Polycomb homologs can
be grouped in two classes (64). Whereas the N-terminal
chromodomains are similar (Fig. 1C), we note that C-terminal homologies
extending beyond the COOH box can serve as another criterion to further distinguish between the two classes of homologs (Fig. 1D). The divergence in the C termini of vertebrate homologs may be of functional significance. Detailed analysis of the molecular basis of Polycomb mutants in Drosophila revealed that many of the mutations
were clustered in the conserved C-terminal domain of Polycomb
(21). The C-terminal domain does not appear to be necessary
for targeting Polycomb to its normal chromosomal binding sites
(21); it is, however, indispensable for transcriptional
repression (9, 44, 64, 66). These observations have led to
the suggestion that the C-terminal domain is important for
protein-protein interactions in recruiting a repressive complex.
Whether the differences in the C-terminal domains of the vertebrate
Polycomb homologs also reflect functional distinctions remains to be seen.
Developmental regulation of XPc1.
The expression analysis of
XPc1 showed high levels of its transcript in the ovary and in immature
Xenopus oocytes, which is strongly suggestive of a maternal
deposition of XPc1 mRNA. Maternal deposition of Pc mRNA has also been
observed in Drosophila oogenesis (51), where
genetic evidence has indeed shown a maternal component in the
developmental functions of Polycomb and other Pc-G members (13,
29, 51). As in Drosophila oocytes (51), we
failed to detect any XPc1 protein in Xenopus oocytes. In
fact, we find that maternally deposited XPc1 Polycomb message is stored
in the oocyte in a translationally inactive form (Fig. 2A). There are features in the XPc1 3' UTR, including the long U tract, that function
as embryonic cytoplasmic polyadenylation elements and are consistent
with the delayed activation of translation (Fig. 2B) (72).
There are interesting parallels between the expression of XPc1 and the
regulation of histone H1 protein synthesis during early
Xenopus development. In this case, masked maternal H1 mRNA is released for translation during the early cleavage divisions, with a
normal stoichiometry of H1 relative to core histones being achieved at
gastrulation (14, 76, 80). The progressive accumulation of
histone H1 leads to the selective repression of oocyte-type 5S rRNA
genes (7, 32) and the loss of mesodermal competence (73, 83). Thus, changes in chromatin composition influence the patterning of the Xenopus embryo into distinct cell lineages.
Transcriptional repression by XPc1.
We find that XPc1 acts as
transcriptional repressor in vivo in Xenopus embryos when
tethered to a promoter by virtue of an N-terminal fusion to the GAL4
DBD (Fig. 3 and 4). Repression by tethering to a promoter has been
reported for many Pc-G members, mostly in transfected cells (2, 9,
63, 64, 66). However, the in vivo repressive effect of tethered
Polycomb during development has been previously described only for
transgenic Drosophila embryos (44). Our results,
therefore, document for the first time the fact that a Polycomb homolog
will also repress transcription during embryonic development in higher
vertebrates. XPc1-mediated repression is first observed shortly after
the MBT stage, when the hsp70 reporter promoter becomes
transcriptionally active. This is consistent with the presence of
GAL4XPc1 protein in the nucleus and suggests that all factors required
for XPc1-mediated repression, for example, other Pc-G members, are also
present in the nucleus at this stage. Repression persisted in all of
the early developmental stages we tested and extended beyond
gastrulation. In Drosophila, repression persists in a
promoter-specific manner even after the GAL4Pc fusion protein has
decayed (44). Presently we do not know whether this is also
the case for our assay in Xenopus embryos, since GAL4XPc1 protein is present in embryonic nuclei in all developmental stages tested.
Using our in vivo repression assay with
Xenopus embryos, we
found that increasing the HSF transcription factor dosage by heat
shock
only partially alleviates XPc1-mediated repression and even
then, hsp70
expression does not reach wild-type, non-heat-shock-induced
levels
(Fig.
4A). This suggests that at least in this reporter
system,
GAL4XPc1 exerts a dominant repressive effect over the
inductive effects
of HSF. This is in contrast to results obtained
with
Drosophila in vivo repression assays. Zink and Paro
(
89)
have used naturally occurring Polycomb response
elements and GAL4
DNA binding sites in transgenic constructs to show
that a large
increase in the amount of the GAL4 transcription factor
can completely
reverse the Pc-G-mediated repression in reporter
constructs. Furthermore,
GAL4 in high doses will also replace Polycomb
binding from reporter
transgenes in polytene chromosomes
(
89). Several factors could
account for the differences
observed between the two assays. For
example, GAL4 may be a more
powerful transcription factor than
HSF, thus completely overcoming
Pc-G-mediated repression. Alternatively,
the GAL4 DNA binding sites
present in our reporter construct may
be more effective than the
Polycomb response element in anchoring
and maintaining GAL4XPc1 to the
promoter.
We established that inhibition of the RPD3 family of histone
deacetylases (
33b) by TSA treatment does not affect
repression
mediated by XPc1 when artificially tethered to a promoter,
thus
suggesting that, at least in this type of assay, deacetylation
is
unlikely to play a role in Polycomb-mediated repression (Fig.
4B).
Control experiments indicate that RPD3-mediated repression
of H1°
gene expression within the endogenous chromosomes of the
Xenopus embryo is relieved by TSA (Fig.
4C and D). It was
recently
postulated that Pc-G repression was mediated by the
recruitment
of histone deacetylases to the Pc-G complex (
32a,
55). Our
results suggest that whatever complex assembles and
mediates the
repression observed in our tethering assay, it does not
involve
an essential deacetylase activity. Ekwall et al. showed
recently
that TSA treatment causes derepression of heterochromatic
silencing
in
S. pombe (
19). TSA treatment also
induces removal from centromeric
heterochromatin of swi6, a
chromodomain-containing HP1-like protein
essential for centromeric
function and a mediator of heterochromatic
silencing (
18,
19). Bearing in mind the parallels that have
often been drawn
between HP1-mediated heterochromatic repression
and Pc-G-mediated
repression, it may be tempting to suggest that,
just like for swi6
silencing in
S. pombe, Pc-G repression also
employs
deacetylation. Our data in fact indicate the opposite,
suggesting that
the molecular bases for heterochromatin silencing
and Pc-G repression
may be different. This is not the first time
that despite the
postulated mechanistic parallels, differences
have been observed at the
molecular level between the two silencing
phenomena. In
Drosophila, transgenes integrated in pericentric
heterochromatin have been suggested to have altered chromatin
structures consistent with a tighter packaging in nucleosomes
(
84). By contrast, Pc-G gene targets in the homeotic loci in
Drosophila did not appear to have altered chromatin
structures,
as evidenced by restriction enzyme accessibility, in the
presence
or absence of Polycomb protein (
65). Alternatively,
the influence
of acetylation on silencing mediated by chromodomain
proteins
may be a phenomenon peculiar to centromeric heterochromatin.
Wallrath
and Elgin (
84) found that genetically induced
histone hyperacetylation,
while derepressing centromeric silenced
domains (
17), did not
affect telomeric silencing, even
though HP1 also appears to be
a component of telomeric heterochromatin.
In that respect, it
would be interesting to test in our assay whether
in
Xenopus embryos
HP1 tethered to a promoter represses
transcription in a TSA-reversible
manner.
By drawing parallels again between the HP1-like swi6 protein and
Polycomb, it was recently proposed that chromodomain proteins
may serve
to faithfully protect and propagate epigenetic states
set up in a
highly localized manner by modifications in chromatin
structure, for
example, deacetylation (
10). According to this
model,
therefore, targeted deacetylation could provide the imprint
for the
establishment of epigenetically regulated transcriptional
states which
will then be maintained by chromodomain protein complexes
through
several mitotic and, surprisingly, meiotic cycles (
10,
11).
Our results cannot distinguish whether deacetylation is
required for
the establishment of repressive states, since tethering
a chromodomain
protein to a promoter bypasses this step altogether.
However, we
presented evidence suggesting that, once established,
deacetylation may
not be involved in the maintenance of repression
mediated by Polycomb
complexes. This may not be the case in HP1
chromodomain-mediated
repression (
19). The presence of a chromodomain
therefore
may signify a common mechanism for specifically targeting
protein
complexes involved in the epigenetic maintenance of transcriptional
states but may not be involved in the molecular mechanism(s) by
which
the effects on transcription are
achieved.
| |
ACKNOWLEDGMENTS |
We are indebted to Nicoletta Landsberger for reagents and
invaluable help.
J.S. has been supported by an HFSPO long-term postdoctoral fellowship.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory of
Molecular Embryology, National Institute of Child Health and Human
Development, National Institutes of Health, Building 18T, Room 106, Bethesda, MD 20892-5431. Phone: (301) 496 4045. Fax: (301) 402 1323. E-mail: awlme{at}helix.nih.gov.
| |
REFERENCES |
| 1.
|
Alkema, M. J.,
M. Bronk,
E. Verhoeven,
A. Otte,
L. J. van't Veer,
A. Berns, and M. van Lohuizen.
1997.
Identification of Bmi1-interacting proteins as constituents of a multimeric mammalian Polycomb complex.
Genes Dev.
11:226-240[Abstract/Free Full Text].
|
| 2.
|
Alkema, M. J.,
J. Jacobs,
J. W. Voncken,
N. A. Jenkins,
N. G. Copeland,
D. P. Satijn,
A. P. Otte,
A. Berns, and M. van Lohuizen.
1997.
MPc2, a new murine homolog of the Drosophila Polycomb protein is a member of the mouse Polycomb transcriptional repressor complex.
J. Mol. Biol.
273:993-1003[Medline].
|
| 3.
|
Almouzni, G., and A. P. Wolffe.
1995.
Constraints on transcriptional activator function contribute to transcriptional quiescence during early Xenopus embryogenesis.
EMBO J.
14:1752-1765[Medline].
|
| 3a.
|
Almouzni, G.,
S. Khochbin,
S. Dimitrov, and A. P. Wolffe.
1994.
Histone acetylation influences both gene expression and development of Xenopus laevis.
Dev. Biol.
165:654-669[Medline].
|
| 4.
|
Andrews, M. T., and D. D. Brown.
1987.
Transient activation of oocyte 5S RNA genes in Xenopus embryos by raising the level of the trans-acting factor TFIIIA.
Cell
51:445-453[Medline].
|
| 5.
|
Bienz, M.
1984.
Xenopus hsp 70 genes are constitutively expressed in injected oocytes.
EMBO J.
3:2477-2483[Medline].
|
| 6.
|
Bienz, M., and J. Müller.
1995.
Transcriptional silencing of homeotic genes in Drosophila.
Bioessays
17:775-784[Medline].
|
| 7.
|
Bouvet, P.,
S. Dimitrov, and A. P. Wolffe.
1994.
Specific regulation of Xenopus chromosomal 5S rRNA gene transcription in vivo by histone H1.
Genes Dev.
8:1147-1159[Abstract/Free Full Text].
|
| 8.
|
Buchenau, P.,
J. Hodgson,
H. Strutt, and D. J. Arndt-Jovin.
1998.
The distribution of Polycomb-group proteins during cell division and development in Drosophila embryos: impact on models for silencing.
J. Cell Biol.
141:469-481[Abstract/Free Full Text].
|
| 9.
|
Bunker, C. A., and R. E. Kingston.
1994.
Transcriptional repression by Drosophila and mammalian Polycomb group proteins in transfected mammalian cells.
Mol. Cell. Biol.
14:1721-1732[Abstract/Free Full Text].
|
| 10.
|
Cavalli, G., and R. Paro.
1998.
Chromo-domain proteins: linking chromatin structure to epigenetic regulation.
Curr. Opin. Cell Biol.
10:354-360[Medline].
|
| 11.
|
Cavalli, G., and R. Paro.
1998.
The Drosophila Fab-7 chromosomal element conveys epigenetic inheritance during mitosis and meiosis.
Cell
93:505-518[Medline].
|
| 12.
|
Davidson, E. H.
1986.
Gene activity in early development.
Academic Press, Inc., Orlando, Fla.
|
| 13.
|
Denell, R. E.
1982.
Homeosis in Drosophila: evidence of a maternal effect of the Polycomb locus.
Dev. Genet.
3:103-113.
|
| 14.
|
Dimitrov, S.,
G. Almouzni,
M. Dasso, and A. P. Wolffe.
1993.
Chromatin transitions during early Xenopus embryogenesis: changes in histone H4 acetylation and in linker histone type.
Dev. Biol.
160:214-227[Medline].
|
| 15.
|
Dombradi, V.,
J. M. Axton,
H. M. Barker, and P. T. Cohen.
1990.
Protein phosphatase 1 activity in Drosophila mutants with abnormalities in mitosis and chromosome condensation.
FEBS Lett.
275:39-43[Medline].
|
| 16.
|
Dombradi, V., and P. T. Cohen.
1992.
Protein phosphorylation is involved in the regulation of chromatin condensation during interphase.
FEBS Lett.
312:21-26[Medline].
|
| 17.
|
Dorn, R.,
S. Heymann,
R. Lindigkeit, and G. Reuter.
1986.
Supressor mutation of position-effect variegation in Drosophila melanogaster affecting chromatin properties.
Chromosoma
93:398-403.
|
| 18.
|
Ekwall, K.,
J. P. Javerzat,
A. Lorentz,
H. Schmidt,
G. Cranston, and R. Allshire.
1995.
The chromodomain protein Swi6: a key component at fission yeast centromeres.
Science
269:1429-1431[Abstract/Free Full Text].
|
| 19.
|
Ekwall, K.,
T. Olsson,
B. M. Turner,
G. Cranston, and R. C. Allshire.
1997.
Transient inhibition of histone deacetylation alters the structural and functional imprint at fission yeast centromeres.
Cell
91:1021-1032[Medline].
|
| 20.
|
Franke, A.,
M. DeCamillis,
D. Zink,
N. Cheng,
H. W. Brock, and R. Paro.
1992.
Polycomb and polyhomeotic are constituents of a multimeric protein complex in chromatin of Drosophila melanogaster.
EMBO J.
11:2941-2950[Medline].
|
| 21.
|
Franke, A.,
S. Messmer, and R. Paro.
1995.
Mapping functional domains of the Polycomb protein of Drosophila melanogaster.
Chromosome Res.
3:351-360[Medline].
|
| 22.
|
Freeman, L.,
H. Kurumizaka, and A. P. Wolffe.
1996.
Functional domains for assembly of histones H3 and H4 into the chromatin of Xenopus embryos.
Proc. Natl. Acad. Sci. USA
93:12780-12785[Abstract/Free Full Text].
|
| 23.
|
Gecz, J.,
S. J. Gaunt,
E. Passage,
R. D. Burton,
C. Cudrey,
J. J. Pearce, and M. Fontes.
1995.
Assignment of a Polycomb-like chromobox gene (CBX2) to human chromosome 17q25.
Genomics
26:130-133[Medline].
|
| 24.
|
Goodrich, J.,
P. Puangsomlee,
M. Martin,
D. Long,
E. M. Meyerowitz, and G. Coupland.
1997.
A Polycomb-group gene regulates homeotic gene expression in Arabidopsis.
Nature
386:44-51[Medline].
|
| 25.
|
Grossniklaus, U.,
J. P. Vielle-Calzada,
M. A. Hoeppner, and W. B. Gagliano.
1998.
Maternal control of embryogenesis by MEDEA, a Polycomb group gene in Arabidopsis.
Science
280:446-450[Abstract/Free Full Text].
|
| 26.
|
Gunster, M. J.,
D. P. Satijn,
K. M. Hamer,
J. L. den Blaauwen,
D. de Bruijn,
M. J. Alkema,
M. van Lohuizen,
R. van Driel, and A. P. Otte.
1997.
Identification and characterization of interactions between the vertebrate polycomb-group protein BMI1 and human homologs of polyhomeotic.
Mol. Cell. Biol.
17:2326-2335[Abstract].
|
| 27.
|
Hair, A.,
M. N. Prioleau,
Y. Vassetzky, and M. Méchali.
1998.
Control of gene expression in Xenopus early development.
Dev. Genet.
22:122-131[Medline].
|
| 28.
|
Hashimoto, N.,
H. W. Brock,
M. Nomura,
M. Kyba,
J. Hodgson,
Y. Fujita,
Y. Takihara,
K. Shimada, and T. Higashinakagawa.
1998.
RAE28, BMI1, and M33 are members of heterogeneous multimeric mammalian Polycomb group complexes.
Biochem. Biophys. Res. Commun.
245:356-365[Medline].
|
| 29.
|
Haynie, J. L.
1983.
The maternal and zygotic roles of the gene Polycomb in embryonic determination in Drosophila melanogaster.
Dev. Biol.
100:399-411[Medline].
|
| 30.
|
Holdeman, R.,
S. Nehrt, and S. Strome.
1998.
MES-2, a maternal protein essential for viability of the germline in Caenorhabditis elegans, is homologous to a Drosophila Polycomb group protein.
Development
125:2457-2467[Abstract].
|
| 31.
|
Jürgens, G.
1985.
A group of genes controlling the spatial expression of the bithorax complex in Drosophila.
Nature
316:153-155.
|
| 32.
|
Kandolf, H.
1994.
The H1A histone variant is an in vivo repressor of oocyte-type 5S gene transcription in Xenopus laevis embryos.
Proc. Natl. Acad. Sci. USA
91:7257-7261[Abstract/Free Full Text].
|
| 32a.
|
Kehle, J.,
D. Benchle,
S. Treuheit,
B. Christen,
J. A. Kennison,
M. Bienz, and J. Muller.
1998.
dMi-2, a Hunchback-interacting protein that functions in Polycomb repression.
Science
282:1897-1900[Abstract/Free Full Text].
|
| 33.
|
Kennison, J. A.
1993.
Transcriptional activation of Drosophila homeotic genes from distant regulatory elements.
Trends Genet.
9:75-79[Medline].
|
| 33a.
|
Khochbin, S., and A. P. Wolffe.
1993.
Developmental regulation and butyrate inducible transcription of the Xenopus histone H1° promoter.
Gene
128:173-180[Medline].
|
| 33b.
|
Khochbin, S., and A. P. Wolffe.
1997.
The origin and utility of histone deacetylases.
FEBS Lett.
419:157-160[Medline].
|
| 34.
|
Koonin, E. V.,
S. Zhou, and J. C. Lucchesi.
1995.
The chromo superfamily: new members, duplication of the chromo domain and possible role in delivering transcription regulators to chromatin.
Nucleic Acids Res.
23:4229-4233[Abstract/Free Full Text].
|
| 35.
|
Korf, I.,
Y. Fan, and S. Strome.
1998.
The Polycomb group in Caenorhabditis elegans and maternal control of germline development.
Development
125:2469-2478[Abstract].
|
| 36.
|
Krone, P. H., and J. J. Heikkila.
1989.
Expression of microinjected hsp 70/CAT and hsp 30/CAT chimeric genes in developing Xenopus laevis embryos.
Development
106:271-281[Abstract].
|
| 37.
|
Landsberger, N.,
M. Ranjan,
G. Almouzni,
D. Stump, and A. P. Wolffe.
1995.
The heat shock response in Xenopus oocytes, embryos, and somatic cells: a regulatory role for chromatin.
Dev. Biol.
170:62-74[Medline].
|
| 38.
|
Landsberger, N., and A. P. Wolffe.
1995.
Role of chromatin and Xenopus laevis heat shock transcription factor in regulation of transcription from the X. laevis hsp70 promoter in vivo.
Mol. Cell. Biol.
15:6013-6024[Abstract].
|
| 39.
|
Landsberger, N., and A. P. Wolffe.
1997.
Remodeling of regulatory nucleoprotein complexes on the Xenopus hsp70 promoter during meiotic maturation of the Xenopus oocyte.
EMBO J.
16:4361-4373[Medline].
|
| 40.
|
Li, Q.,
M. Herrler,
N. Landsberger,
N. Kaludov,
V. V. Ogryzko,
Y. Nakatani, and A. P. Wolffe.
1998.
Xenopus NF-Y pre-sets chromatin to potentiate p300 and acetylation-responsive transcription from the Xenopus hsp70 promoter in vivo.
EMBO J.
17:6300-6315[Medline].
|
| 41.
|
Meric, F.,
K. Matsumoto, and A. P. Wolffe.
1997.
Regulated unmasking of in vivo synthesized maternal mRNA at oocyte maturation. A role for the chaperone nucleoplasmin.
J. Biol. Chem.
272:12840-12846[Abstract/Free Full Text].
|
| 42.
|
Messmer, S.,
A. Franke, and R. Paro.
1992.
Analysis of the functional role of the Polycomb chromo domain in Drosophila melanogaster.
Genes Dev.
6:1241-1254[Abstract/Free Full Text].
|
| 43.
|
Moehrle, A., and R. Paro.
1994.
Spreading the silence: epigenetic transcriptional regulation during Drosophila development.
Dev. Genet.
15:478-484[Medline].
|
| 44.
|
Müller, J.
1995.
Transcriptional silencing by the Polycomb protein in Drosophila embryos.
EMBO J.
14:1209-1220[Medline].
|
| 45.
|
Müller, J.,
S. Gaunt, and P. A. Lawrence.
1995.
Function of the Polycomb protein is conserved in mice and flies.
Development
121:2847-2852[Abstract].
|
| 46.
|
Orlando, V., and R. Paro.
1993.
Mapping Polycomb-repressed domains in the bithorax complex using in vivo formaldehyde cross-linked chromatin.
Cell
75:1187-1198[Medline].
|
| 47.
|
Paro, R.
1990.
Imprinting a determined state into the chromatin of Drosophila.
Trends Genet.
6:416-421[Medline].
|
| 48.
|
Paro, R.
1993.
Mechanisms of heritable gene repression during development of Drosophila.
Curr. Opin. Cell Biol.
5:999-1005[Medline].
|
| 49.
|
Paro, R., and P. J. Harte.
1996.
The role of Polycomb Group and Trithorax Group chromatin complexes in the maintenance of determined cells rates, p. 507-528.
In
V. E. A. Russo, R. A. Martienssen, and A. D. Riggs (ed.), Epigenetic mechanisms of gene regulation. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 50.
|
Paro, R., and D. S. Hogness.
1991.
The Polycomb protein shares a homologous domain with a heterochromatin-associated protein of Drosophila.
Proc. Natl. Acad. Sci. USA
88:263-267[Abstract/Free Full Text].
|
| 51.
|
Paro, R., and B. Zink.
1993.
The Polycomb gene is differentially regulated during oogenesis and embryogenesis of Drosophila melanogaster.
Mech. Dev.
40:37-46[Medline].
|
| 52.
|
Parthun, M. R., and J. A. Jaehning.
1992.
A transcriptionally active form of GAL4 is phosphorylated and associated with GAL80.
Mol. Cell. Biol.
12:4981-4987[Abstract/Free Full Text].
|
| 53.
|
Pearce, J. J.,
P. B. Singh, and S. J. Gaunt.
1992.
The mouse has a Polycomb-like chromobox gene.
Development
114:921-929[Abstract].
|
| 54.
|
Pirrotta, V.
1997.
PcG complexes and chromatin silencing.
Curr. Opin. Genet. Dev.
7:249-258[Medline].
|
| 55.
|
Pirrotta, V.
1998.
Polycombing the genome: PcG, trxG, and chromatin silencing.
Cell
93:333-336[Medline].
|
| 56.
|
Pirrotta, V., and L. Rastelli.
1994.
White gene expression, repressive chromatin domains and homeotic gene regulation in Drosophila.
Bioessays
16:549-556[Medline].
|
| 57.
|
Prioleau, M. N.,
J. Huet,
A. Séntenac, and M. Méchali.
1994.
Competition between chromatin and transcription complex assembly regulates gene expression during early development.
Cell
77:439-449[Medline].
|
| 58.
|
Prioleau, M. N.,
R. S. Buckle, and M. Méchali.
1995.
Programming of a repressed but committed chromatin structure during early development.
EMBO J.
14:5073-5084[Medline].
|
| 59.
|
Ranjan, M.,
S. R. Tafuri, and A. P. Wolffe.
1993.
Masking mRNA from translation in somatic cells.
Genes Dev.
7:1725-1736[Abstract/Free Full Text].
|
| 60.
|
Rastelli, L.,
C. S. Chan, and V. Pirrotta.
1993.
Related chromosome binding sites for zeste, suppressors of zeste and Polycomb group proteins in Drosophila and their dependence on Enhancer of zeste function.
EMBO J.
12:1513-1522[Medline].
|
| 61.
|
Reijnen, M. J.,
K. M. Hamer,
J. L. den Blaauwen,
C. Lambrechts,
I. Schoneveld,
R. van Driel, and A. P. Otte.
1995.
Polycomb and bmi-1 homologs are expressed in overlapping patterns in Xenopus embryos and are able to interact with each other.
Mech. Dev.
53:35-46[Medline].
|
| 62.
|
Sargent, T. D., and P. H. Mathers.
1991.
Analysis of class II gene regulation.
Methods Cell Biol.
36:347-364[Medline].
|
| 63.
|
Satijn, D. P.,
M. J. Gunster,
J. van der Vlag,
K. M. Hamer,
W. Schul,
M. J. Alkema,
A. J. Saurin,
P. S. Freemont,
R. van Driel, and A. P. Otte.
1997.
RING1 is associated with the Polycomb group protein complex and acts as a transcriptional repressor.
Mol. Cell. Biol.
17:4105-4113[Abstract].
|
| 64.
|
Satijn, D. P.,
D. J. Olson,
J. van der Vlag,
K. M. Hamer,
C. Lambrechts,
H. Masselink,
M. J. Gunster,
R. G. Sewalt,
R. van Driel, and A. P. Otte.
1997.
Interference with the expression of a novel human Polycomb protein, hPc2, results in cellular transformation and apoptosis.
Mol. Cell. Biol.
17:6076-6086[Abstract].
|
| 65.
|
Schlossherr, J.,
H. Eggert,
R. Paro,
S. Cremer, and R. S. Jack.
1994.
Gene inactivation in Drosophila mediated by the Polycomb gene product or by position-effect variegation does not involve major changes in the accessibility of the chromatin fibre.
Mol. Gen. Genet.
243:453-462[Medline].
|
| 66.
|
Schoorlemmer, J.,
C. Marcos-Gutierrez,
F. Were,
R. Martinez,
E. Garcia,
D. P. Satijn,
A. P. Otte, and M. Vidal.
1997.
Ring1A is a transcriptional repressor that interacts with the Polycomb-M33 protein and is expressed at rhombomere boundaries in the mouse hindbrain.
EMBO J.
16:5930-5942[Medline].
|
| 67.
|
Schumacher, A., and T. Magnuson.
1997.
Murine Polycomb- and trithorax-group genes regulate homeotic pathways and beyond.
Trends Genet.
13:167-170.
|
| 68.
|
Schumacher, A.,
C. Faust, and T. Magnuson.
1996.
Positional cloning of a global regulator of anterior-posterior patterning in mice.
Nature
384:648[Medline].
|
| 69.
|
Sewalt, R. G.,
J. van der Vlag,
M. J. Gunster,
K. M. Hamer,
J. L. den Blaauwen,
D. P. Satijn,
T. Hendrix,
R. van Driel, and A. P. Otte.
1998.
Characterization of interactions between the mammalian Polycomb-group proteins Enx1/EZH2 and EED suggests the existence of different mammalian Polycomb-group protein complexes.
Mol. Cell. Biol.
18:3586-3595[Abstract/Free Full Text].
|
| 70.
|
Simon, J.,
A. Chiang, and W. Bender.
1992.
Ten different Polycomb group genes are required for spatial control of the AbdA and AbdB homeotic products.
Development
114:493-505[Abstract].
|
| 71.
|
Smith, L. D.,
W. Xu, and R. L. Varnold.
1991.
Oogenesis and oocyte isolation.
Methods Cell Biol.
36:45-60[Medline].
|
| 72.
|
Stebbins-Boaz, B., and J. D. Richter.
1997.
Translational control during early development.
Crit. Rev. Eukaryot. Gene Expr.
7:73-94[Medline].
|
| 73.
|
Steinbach, O. C.,
A. P. Wolffe, and R. A. Rupp.
1997.
Somatic linker histones cause loss of mesodermal competence in Xenopus.
Nature
389:395-399[Medline].
|
| 74.
|
Strutt, H.,
G. Cavalli, and R. Paro.
1997.
Co-localization of Polycomb protein and GAGA factor on regulatory elements responsible for the maintenance of homeotic gene expression.
EMBO J.
16:3621-3632[Medline].
|
| 75.
|
Stump, D. G.,
N. Landsberger, and A. P. Wolffe.
1995.
The cDNA encoding Xenopus laevis heat-shock factor 1 (XHSF1): nucleotide and deduced amino-acid sequences, and properties of the encoded protein.
Gene
160:207-211[Medline].
|
| 76.
|
Tafuri, S. R., and A. P. Wolffe.
1993.
Selective recruitment of masked maternal mRNA from messenger ribonucleoprotein particles containing FRGY2 (mRNP4).
J. Biol. Chem.
268:24255-24261[Abstract/Free Full Text].
|
| 77.
|
Takihara, Y.,
D. Tomotsune,
M. Shirai,
Y. Katoh-Fukui,
K. Nishii,
M. A. Motaleb,
M. Nomura,
R. Tsuchiya,
Y. Fujita,
Y. Shibata,
T. Higashinakagawa, and K. Shimada.
1997.
Targeted disruption of the mouse homologue of the Drosophila polyhomeotic gene leads to altered anteroposterior patterning and neural crest defects.
Development
124:3673-3682[Abstract].
|
| 78.
|
Tamkun, J. W.
1995.
The role of brahma and related proteins in transcription and development.
Curr. Opin. Genet. Dev.
5:473-477[Medline].
|
| 79.
|
Tamkun, J. W.,
R. Deuring,
M. P. Scott,
M. Kissinger,
A. M. Pattatucci,
T. C. Kaufman, and J. A. Kennison.
1992.
brahma: a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2.
Cell
68:561-572[Medline].
|
| 80.
|
Toyoda, T., and A. P. Wolffe.
1992.
Characterization of RNA polymerase II-dependent transcription in Xenopus extracts.
Dev. Biol.
153:150-157[Medline].
|
| 81.
|
Tyers, M.,
G. Tokiwa,
R. Nash, and B. Futcher.
1992.
The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation.
EMBO J.
11:1773-1784[Medline].
|
| 82.
|
van Lohuizen, M.,
M. Tijms,
J. W. Voncken,
A. Schumacher,
T. Magnuson, and E. Wientjens.
1998.
Interaction of mouse Polycomb-group (Pc-G) proteins Enx1 and Enx2 with Eed: indication for separate Pc-G complexes.
Mol. Cell. Biol.
18:3572-3579[Abstract/Free Full Text].
|
| 83.
|
Vermaak, D.,
O. C. Steinbach,
S. Dimitrov,
R. A. W. Rupp, and A. P. Wolffe.
1998.
The globular domain of histone H1 is sufficient to direct specific gene repression in early Xenopus embryos.
Curr. Biol.
23:533-536.
|
| 83a.
| Wade, P. Personal communication.
|
| 83b.
|
Wade, P. A.,
P. L. Jones,
D. Vermaak, and A. P. Wolffe.
1998.
The multiple subunit Mi-2 histone deacetylase from Xenopus laevis contains a Snf2 superfamily ATPase.
Curr. Biol.
8:843-846[Medline].
|
| 84.
|
Wallrath, L. L., and S. C. Elgin.
1995.
Position effect variegation in Drosophila is associated with an altered chromatin structure.
Genes Dev.
9:1263-1277[Abstract/Free Full Text].
|
| 85.
|
Wedeen, C.,
K. Harding, and M. Levine.
1986.
Spatial regulation of Antennapedia and bithorax gene expression by the Polycomb locus in Drosophila.
Cell
44:739-748[Medline].
|
| 86.
|
Wong, J.,
D. Patterton,
A. Imhof,
D. Guschin,
Y.-B. Shi, and A. P. Wolffe.
1998.
Distinct requirements for chromatin assembly in transcriptional repression by thyroid hormone receptor and histone deacetylase.
EMBO J.
17:520-534[Medline].
|
| 86a.
|
Woodland, H. R.,
J. M. Flynn, and A. J. Wyllie.
1979.
Utilization of stored mRNA in Xenopus embryos and its replacement by newly synthesized transcripts: histone H1 synthesis using interspecies hybrids.
Cell
18:165-171[Medline].
|
| 87.
|
Yamaguchi, K.,
S. Hidema, and S. Mizuno.
1998.
Chicken chromobox proteins: cDNA cloning of CHCB1, -2, -3 and their relation to W-heterochromatin.
Exp. Cell Res.
242:303-314[Medline].
|
| 88.
|
Zink, B., and R. Paro.
1989.
In vivo binding pattern of a trans-regulator of homeotic genes in Drosophila melanogaster.
Nature
337:468-471[Medline].
|
| 89.
|
Zink, B., and R. Paro.
1995.
Drosophila Polycomb-group regulated chromatin inhibits the accessibility of a trans-activator to its target DNA.
EMBO J.
14:5660-5671[Medline].
|
Molecular and Cellular Biology, June 1999, p. 3958-3968, Vol. 19, No. 6
0270-7306/99/$04.00+0
This article has been cited by other articles:
-
Kelly, R. G., Lemonnier, M., Zaffran, S., Munk, A., Buckingham, M. E.
(2003). Cell history determines the maintenance of transcriptional differences between left and right ventricular cardiomyocytes in the developing mouse heart. J. Cell Sci.
116: 5005-5013
[Abstract]
[Full Text]
-
Hur, M.-W., Laney, J. D., Jeon, S.-H., Ali, J., Biggin, M. D.
(2003). Zeste maintains repression of Ubx transgenes: support for a new model of Polycomb repression. Development
129: 1339-1343
[Abstract]
[Full Text]
-
Mohd-Sarip, A., Venturini, F., Chalkley, G. E., Verrijzer, C. P.
(2002). Pleiohomeotic Can Link Polycomb to DNA and Mediate Transcriptional Repression. Mol. Cell. Biol.
22: 7473-7483
[Abstract]
[Full Text]
-
Chang, Y.-L., Peng, Y.-H., Pan, I-C., Sun, D.-S., King, B., Huang, D.-H.
(2001). Essential role of Drosophila Hdac1 in homeotic gene silencing. Proc. Natl. Acad. Sci. USA
10.1073/pnas.171325498v1
[Abstract]
[Full Text]
-
Tussie-Luna, M. I., Bayarsaihan, D., Ruddle, F. H., Roy, A. L.
(2001). Repression of TFII-I-dependent transcription by nuclear exclusion. Proc. Natl. Acad. Sci. USA
98: 7789-7794
[Abstract]
[Full Text]
-
Wen, X., Wu, G. D.
(2001). Evidence for Epigenetic Mechanisms That Silence Both Basal and Immune-Stimulated Transcription of the IL-8 Gene. J. Immunol.
166: 7290-7299
[Abstract]
[Full Text]
-
Izutsu, K., Kurokawa, M., Imai, Y., Maki, K., Mitani, K., Hirai, H.
(2001). The corepressor CtBP interacts with Evi-1 to repress transforming growth factor {beta} signaling. Blood
97: 2815-2822
[Abstract]
[Full Text]
-
Satijn, D. P. E., Hamer, K. M., den Blaauwen, J., Otte, A. P.
(2001). The Polycomb Group Protein EED Interacts with YY1, and Both Proteins Induce Neural Tissue in Xenopus Embryos. Mol. Cell. Biol.
21: 1360-1369
[Abstract]
[Full Text]
-
Akasaka, T, van Lohuizen, M, van der Lugt, N, Mizutani-Koseki, Y, Kanno, M, Taniguchi, M, Vidal, M, Alkema, M, Berns, A, Koseki, H
(2001). Mice doubly deficient for the Polycomb Group genes Mel18 and Bmi1 reveal synergy and requirement for maintenance but not initiation of Hox gene expression. Development
128: 1587-1597
[Abstract]
-
Kikyo, N, Wolffe, A.
(2000). Reprogramming nuclei: insights from cloning, nuclear transfer and heterokaryons. J. Cell Sci.
113: 11-20
[Abstract]
-
Koipally, J., Georgopoulos, K.
(2000). Ikaros Interactions with CtBP Reveal a Repression Mechanism That Is Independent of Histone Deacetylase Activity. J. Biol. Chem.
275: 19594-19602
[Abstract]
[Full Text]
-
Chang, Y.-L., Peng, Y.-H., Pan, I-C., Sun, D.-S., King, B., Huang, D.-H.
(2001). Essential role of Drosophila Hdac1 in homeotic gene silencing. Proc. Natl. Acad. Sci. USA
98: 9730-9735
[Abstract]
[Full Text]
Top
Abstract
Introduction
