|
|
|
|
|
|
|
||||
| Home Help Feedback Subscriptions Archive Search Table of Contents | ||||
First published online 13 March 2008
doi: 10.1242/dev.014340
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
1 RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku,
Yokohama 230-0045, Japan.
2 RIKEN Genomic Sciences Center, 1-7-22 Suehiro, Tsurumi-ku, Yokohama 230-0045,
Japan.
3 Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan
406, 1098 SM Amsterdam, The Netherlands.
4 RIKEN Center for Developmental Biology, 2-2-3 Minatojima-minamimachi, Chuo-ku,
Kobe, Hyogo 6500047, Japan.
5 Developmental Epigenetics Group, MRC Clinical Sciences Centre, ICFM,
Hammersmith Hospital, DuCane Road, London W12 ONN, UK.
6 Centro de Investigaciones Biologicas, Department of Developmental and Cell
Biology, Ramiro de Maeztu 9, 28040 Madrid, Spain.
* Author for correspondence (e-mail: koseki{at}rcai.riken.jp)
Accepted 29 January 2008
 |
SUMMARY |
|---|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Key words: Polycomb, Oct3/4 (Pou5f1), Gata6, ES cells, Chromatin, Silencing, Ring1A/B (Ring1/Rnf2), Mouse
|
INTRODUCTION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Martin,
1981). These properties of ES cells are maintained by symmetrical
self-renewal, producing two identical stem cell daughters upon cell division
(Burdon et al., 2002). The
maintenance of ES cell pluripotency is thought to involve a transcriptional
regulatory hierarchy including the transcription factors Oct3/4 (Pou5f1 -
Mouse Genome Informatics), Sox2 and Nanog, which may be central components
judging by their unique expression patterns and essential roles during early
murine development (Avilion et al.,
2003; Chambers et al.,
2003; Mitsui et al.,
2003; Nichols et al.,
1998).
Recent genome-wide chromatin immunoprecipitation (ChIP) analyses revealed
that OCT4, SOX2 and NANOG co-occupy the promoters of a large group of genes in
human ES cells (Boyer et al.,
2005), suggesting that these factors form a core regulatory
feedback circuit, in which all three factors regulate the expression of
themselves as well as of each other
(Catena et al., 2004;
Kuroda et al., 2005;
Okumura-Nakanishi et al.,
2005; Rodda et al.,
2005). This positive-feedback loop promotes self-renewal of
pluripotent ES cells by repressing transcription factors involved in
differentiation and development, whilst likely activating the expression of
genes involved in ES cell maintenance
(Boyer et al., 2005).
The execution of differentiation programs in ES cells is likely to be
preceded by interruption of the positive-feedback loop by developmental
regulators such as Cdx2 and Gata6. The expression of Cdx2 and Gata6 is
repressed by Oct3/4 and Nanog in undifferentiated ES cells
(Boyer et al., 2005;
Loh et al., 2006;
Mitsui et al., 2003;
Niwa et al., 2000;
Niwa et al., 2005), and the
enforced expression of Cdx2 and Gata6 quickly shuts down the positive loop and
promotes a rapid transition from the undifferentiated to the differentiated
state (Fujikura et al., 2002;
Niwa et al., 2005). Therefore,
the positive-feedback loops and developmental regulators are reciprocally
engaged to maintain ES cell identity; however, the molecular mechanisms
underlying this reciprocal interaction are not fully understood.
The Polycomb group (PcG) of proteins mediate heritable silencing of
developmental regulators in metazoans, participating in one of two distinct
multimeric protein complexes, the Polycomb repressive complexes 1 (PRC1) and 2
(PRC2) (Cao et al., 2002;
Czermin et al., 2002;
Kuzmichev et al., 2002;
Muller et al., 2002;
Shao et al., 1999). In
mammals, the core PRC2 is composed of Eed, Ezh2 and Suz12 and catalyses
trimethylation of histone H3 at lysine 27 (H3K27), which in turn is thought to
provide a recruitment site for PRC1 (Cao
et al., 2002; Czermin et al.,
2002; Fischle et al.,
2003; Kuzmichev et al.,
2002; Min et al.,
2003). The core PRC1 is composed of orthologs of
Drosophila Polycomb (Cbx2, Cbx4 and Cbx8), Posterior sex combs [Mel18
(Pcgf2) and Bmi1], Sex comb extra (Ring1A and Ring1B, also known as Ring1 and
Rnf2, respectively - Mouse Genome Informatics) and Polyhomeotic (Phc1, Phc2
and Phc3). Recent studies demonstrate that mono-ubiquitylation of histone H2A
at lysine 119 is important in PcG-mediated silencing, with Ring1A/B
functioning as the E3 ligase in this reaction
(de Napoles et al., 2004;
Wang et al., 2004).
Mouse and human ES cells have recently been analyzed by genome-wide ChIP,
and PRC1 and PRC2 have been shown to repress genes involved in processes
including development, transcriptional regulation and morphogenesis, via
direct interactions with target genes
(Boyer et al., 2006;
Lee et al., 2006). Notably,
PRC2 has been shown to share target genes with OCT4, SOX2 and/or NANOG in
human ES cells (Lee et al.,
2006). However, it is still unclear whether PcG and the core
transcription network are functionally linked to regulate expression of their
target genes.
In this study, we addressed the role of PRC1 in mouse ES cell maintenance and its functional interaction with the core transcriptional regulatory circuitry. We find that PRC1 is essential for the maintenance of ES cell identity and for the repression of developmental regulators by inhibiting chromatin remodeling. We go on to show that Ring1A/B-mediated PcG silencing is Oct3/4-dependent, whereas it is abolished by developmental cues resulting in Gata6 activation. Collectively, our data suggest that Ring1A/B-mediated Polycomb silencing functions downstream of the core transcriptional regulatory circuitry to maintain ES cell self-renewal.
|
MATERIALS AND METHODS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
; Lei et al.,
1996; Niwa et al.,
2000; Shimosato et al.,
2007). Generation of mutant Ring1A and Ring1B
flox alleles were described previously
(Cales et al., 2008;
del Mar Lorente et al., 2000).
Rosa26::CreERT2 transgenic mice were purchased from Artemis
Pharmaceuticals (Seibler et al.,
2003).
Ring1A-/-;Ring1Bfl/fl;
Rosa26::CreERT2 ES cells were derived from blastocysts. Male
ES cells were used in this study.
Immunoprecipitation (IP) and chromatin immunoprecipitation (ChIP) analyses
IP (Isono et al., 2005a)
and ChIP (Orlando et al.,
1997) were performed as previously described. Immunoprecipitated
and input DNA were quantified by real-time PCR. Primer and probe sequences are
available upon request. Antibodies used in this study are listed in
Table 1.
|
).
Quantitative real-time PCR was carried out using the SYBR Green (Stratagene,
Agilent Technologies, Santa Clara, CA) or Taqman (Biosearch Technologies,
Novato, CA) method and amplifications detected with an Mx3005P (Stratagene, La
Jolla, CA). Primer and probe sequences are available upon request.
Microarray methods and data analysis
Total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA) and
purified with RNeasy separation columns (Qiagen, Hilden, Germany).
First-strand cDNA was synthesized and hybridized to Affymetrix GeneChip Mouse
Genome 430 2.0 arrays (Affymetrix, Santa Clara, CA) to assess and compare the
overall gene expression profiles.
To obtain normalized intensities from at least two slides, the quantile
normalization method was used for every feature on the array
(Bolstad et al., 2003). We
calculated the log of the ratio of intensity in the knockout (KO) samples to
the intensity in the respective control samples. Probes were not applied for
further analysis when signals were at insignificant levels in control and KO
samples. The expression change of a gene was calculated using the geometric
mean of all probes aligned on the gene.
The microarray and ChIP-chip data are available in the NCBI Gene Expression Omnibus (GEO) under the series GSE10573 [NCBI GEO] with sample accession numbers GSM265040 to GSM265045, GSM266065 to GSM266067, GSM266076, GSM266077, GSM266115, GSM266837 and GSM266838.
Comparable expression analyses between KO ES cells
We obtained Pearson product-moment correlation coefficients of the
logarithms of expression changes between respective KO ES cells. The 95%
confidence intervals of correlation coefficients were calculated using Z
transformation. Eigenvalues and eigenvectors of the distribution in scatter
diagrams were calculated using principal component analysis with software R
(http://www.r-project.org/).
Gene ontology (GO) analysis
We performed GO analysis using our in-house programs written in Python and
C++ and GO data retrieved from the Gene Ontology database
(http://www.geneontology.org),
KEGG
(http://www.genome.jp/kegg/)
and others (Auernhammer and Melmed,
2000; Heinrich et al.,
2003). The version of the dataset used was Oct 27th, 2006,
submitted by Mouse Genome Informatics (MGI). We aligned microarray probes on
mouse genes and assigned GO terms on all probes using these alignments. The
significance of each GO term was determined using Fisher's exact test and
Bonferroni adjustment for multiple testing. The P-value reflects the
likelihood that we would observe such enrichment or higher by chance.
Subsequent statistical examinations were also conducted using Fisher's exact
test.
ChIP-chip experiment, assignment of IP regions and calculation of fold enrichment
ChIP-on-chip analysis of Ring1B binding was carried out using the Mouse
Promoter ChIP-on-chip Microarray Set (G4490A; Agilent Technologies). ES cells
were subjected to ChIP assay using anti-Ring1B antibody as described
(Fujimura et al., 2006).
Purified immunoprecipitated and input DNA were subjected to blunt ligation
with linker oligo DNA, linker-mediated PCR (LM-PCR), labeling, hybridization
and washing following the Agilent mammalian ChIP-on-chip protocol. Scanned
images were quantified with Agilent Feature Extraction software under standard
conditions.
Assignment of regions bound by Ring1B around transcription start sites (TSSs) was carried out using direct sequence alignment on the mouse genome database (NCBI version 36). The location of Ring1B-bound regions was compared with a set of transcripts derived from the MGI database. We assigned bound regions that were within -8 kb to +2 kb of the TSS. Alignments on mouse genome and TSSs of genes were retrieved from Ensembl (http://www.ensembl.org).
The measured intensity ratios (IP/input: fold enrichment) were calculated, and the maximum value of the ratios in each promoter region (-8 kb to +2 kb around TSS) of a gene was used to represent the fold enrichment of the gene. Fold enrichment was calculated only for probes whose signals both from IP and input DNAs were significant (P<10-3).
|
RESULTS |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
;
Fujimura et al., 2006) (data
not shown). Thus, we established
Ring1A-/-;Ring1Bfl/fl;
Rosa26::CreERT2 ES cell lines, in which Ring1B
could be conditionally deleted by 4-hydroxy tamoxifen (OHT) treatment. Ring1B
protein levels were dramatically depleted within 48 hours of OHT
administration (Fig. 1A), and
loss of Ring1B resulted in reduced levels of other PRC1 components Mel18,
Phc1/2 and Cbx2 (Fujimura et al.,
2006; Leeb and Wutz,
2007). At a global level, PRC1-regulated mono-ubiquitylated
histone H2A (H2Aub1) was rapidly depleted within 48 hours following OHT
treatment (Fig. 1B). We thus
concluded that PRC1 could be conditionally depleted in this ES cell line by
OHT.
In the Ring1A/B-dKO, in contrast to the Ring1B-KO ES
cells, proliferation was halted and the cells gradually lost typical ES cell
morphology after OHT administration (Fig.
1C). Moreover, genome-wide mRNA analysis revealed preferential
derepression of genes involved in differentiation and/or developmental
processes (Fig. 1D; see also
Table S1 in the supplementary material). These observations, considered
together with structural and biochemical similarities of Ring1A and Ring1B
(Buchwald et al., 2006), led
us to hypothesize a compensatory role of Ring1A for Ring1B in the repression
of developmental genes in ES cells. This idea is partly supported by the
increased expression of Ring1A protein observed in the Ring1B-KO ES
cells (see Fig. S1 in the supplementary material). We first compared gene
expression between the Ring1A/B-dKO and Ring1B-KO ES cells
by microarray analyses. We found that 491 genes were derepressed more than
2-fold in Ring1B-KO (constitutive), whereas in Ring1A/B-dKO
(day 4) ES cells, 999 genes were derepressed (see Table S2 in the
supplementary material). Fold expression changes for respective probes in
Ring1A/B-dKO and Ring1B-KO ES cells, determined against the
parental or wild-type cells, were plotted on a scatter diagram and the
correlation was calculated according to Pearson (see Fig. S2 in the
supplementary material). We found a strong correlation (r=0.386) in
total calculable genes (see Fig. S2 in the supplementary material). This
result indicates significant overlap of genes derepressed in
Ring1A/B-dKO and Ring1B-KO ES cells. The level of
derepression was much higher in Ring1A/B-dKO than in
Ring1B-KO ES cells, as represented by differences in variance (see
Fig. S2 in the supplementary material). On average, developmental genes were
1.389-fold derepressed in Ring1A/B-dKO ES cells, but 1.046-fold in
Ring1B-KO (see Table S3 in the supplementary material). We confirmed
these quantitative differences by evaluating the expression levels of several
developmental regulators including Gata6 and Cdx2 by
quantitative RT-PCR. These genes were significantly derepressed by conditional
depletion of Ring1B, but the degree of derepression was higher in the
Ring1A/B-dKO than in the Ring1B-KO cells
(Fig. 1E). Therefore, Ring1A
and Ring1B appear to act in a compensatory manner to repress the expression of
developmental regulators in ES cells and consequently contribute to the
maintenance of ES cells in an undifferentiated state. The phenotypic
differences between Ring1B-KO and Ring1A/B-dKO ES cells are
likely to be due to exaggerated derepression of developmental regulators such
as Gata6 and Cdx2 in the dKO cells.
We next examined whether the derepression of developmental regulators is accompanied by disruption of the core transcriptional regulatory circuitry in ES cells. We performed multicolor immunofluorescence analysis for Oct3/4 and for Gata4, which is also expressed in primitive endoderm. Oct3/4 was expressed relatively uniformly in all of the control cells and Gata4 was also expressed in most of the cells (Fig. 1F, upper panels). Four days after OHT administration, we found striking heterogeneity in Oct3/4 expression (Fig. 1F, lower panels). In the example illustrated, most of the cells are compacted; however, a subset of cells at the edge spread out from the colony and exhibit epithelial cell morphology. Most of these cells expressed Gata4 but not Oct3/4 (Fig. 1F, arrowheads in lower right panel), which is likely to be indicative of the onset of spontaneous differentiation. Taken together with the gene expression analysis, these results indicate that Ring1A/B contribute crucially to the repression of ES cell differentiation and therefore to the maintenance of ES cell identity.
Ring1A/B mediate repression of developmental regulators by inhibiting chromatin remodeling via direct binding
We next used ChIP analysis to determine whether genes derepressed in
Ring1A/B-dKO ES cells were direct targets of PRC1. As shown in
Fig. 2A, we observed binding of
Ring1B and Phc1, another component of PRC1, to Hoxb8, Gata6, Cdx2,
Zic1 and T, all of which are derepressed in the
Ring1A/B-dKO (Fig.
1E). Binding to all these genes was significantly reduced 2 days
after administration of OHT in
Ring1A-/-;Ring1Bfl/fl;Rosa26::CreERT2
ES cells, suggesting that Ring1B is essential for the establishment of PRC1 at
their respective loci.
We next examined whether genes derepressed in Ring1A/B-dKO ES
cells were bound by Ring1B at their promoters using a ChIP-chip approach
(Fig. 2B; see Tables S4 and S5
in the supplementary material). We identified almost the same set of Ring1B
targets that had been reported previously, if a certain threshold is adopted
to distinguish genes bound by Ring1B (Boyer
et al., 2006) (data not shown). We further clarified linear
correlations between the degree of Ring1B binding and derepression in
Ring1B-KO and Ring1A/B-dKO ES cells
(Fig. 2B). These results
indicate that Ring1A/B generally repress transcription by directly binding to
the target loci in a dose-dependent manner.
Recent studies have demonstrated that PcG targets in ES cells are often
characterized by a unique chromatin configuration, being simultaneously
enriched for histone modifications associated with gene activity [histone H3
lysine 4 trimethylation (H3K4me3) and lysine 9/14 acetylation (H3Ac)] and
modifications associated with PcG-mediated repression [specifically H3K27
trimethylation (H3K27me3)] (Azuara et al.,
2006; Bernstein et al.,
2006). With this in mind, we investigated changes in chromatin
configuration upon Ring1A/B depletion. At a global level, PRC1-mediated H2Aub1
was rapidly depleted (Fig. 1B).
By contrast, there was no detectable change in overall levels of either
H3K4me3, H3Ac, H3K27me3 or PRC2 components (see Fig. S3 in the supplementary
material). We then analyzed promoter regions of selected PcG target loci
derepressed in Ring1A/B-dKO ES cells by ChIP. In addition to histone
modifications, we analyzed binding of Eed and non-phosphorylated RNA
polymerase II (RNAPII) (Fig.
2C). Levels of H3Ac, H3K4me3 and RNAPII binding were significantly
increased, whereas those of Eed and H3K27me3 were decreased. Although the
molecular mechanism for the decrease in Eed binding upon Ring1A/B depletion is
unclear, it is possible that changes in chromatin structure caused by Ring1A/B
depletion might secondarily affect Eed binding.
|
;
Bernstein et al., 2006).
A large number of genes are repressed by both Ring1A/B and Oct3/4
Given that Ring1A/B are required for the maintenance of ES cell identity,
we next examined the relationship between Ring1A/B and the core
transcriptional regulatory circuitry in ES cells, because a previous study
demonstrated that OCT3/4, SOX2 and NANOG co-occupy a significant subset of
PRC2 target genes in human ES cells (Lee
et al., 2006). In fact, inactive genes bound by OCT3/4, SOX2
and/or NANOG in human ES cells are overrepresented among those genes more than
2-fold derepressed in Ring1A/B-dKO mouse ES cells (see Fig. S4 in the
supplementary material).
To directly test whether Ring1A/B mediate transcriptional silencing by the
core transcriptional circuitry we made use of Oct3/4 conditional
knockout ES cells (ZHBTc4) (Niwa et al.,
2000), comparing changes in gene expression in Oct3/4-KO
and Ring1A/B-dKO cells. Because most Oct3/4-KO ES cells
begin to exhibit trophectoderm-like morphology within 2 to 3 days after
induction (Niwa et al., 2000),
we analyzed RNA from ES cells 1 day after tetracycline (Tc) treatment, at
which time Oct3/4 protein is extensively depleted, thus minimizing the
contribution of secondary changes in gene expression resulting from
differentiation. As controls, we also analyzed gene expression in
Eed- and Dnmt1-KO ES cells. Dnmt1-KO ES cells
self-renew but fail to undergo differentiation upon induction
(Lei et al., 1996). Fold
changes for respective probes determined against the parental cells were
distributed on scatter diagrams and the correlation among respective KO ES
cells was calculated (Fig. 3A).
We found a strong correlation in total calculable genes between
Oct3/4-KO and Ring1A/B-dKO ES cells (r=0.279).
Overall gene expression in Ring1A/B-dKO ES cells also exhibited a
strong correlation (r=0.359) with the Eed-KO, which might
represent functional engagement of PRC1 and PRC2. By contrast, we found no
correlation of the Dnmt1-KO with either the Ring1A/B-dKO
(r=0.078) or Oct3/4-KO (r=-0.001). This analysis
indicates that a large number of genes in ES cells are concurrently repressed
by Oct3/4 and Ring1A/B.
Next we tested which genes regulated by Oct3/4 and Ring1A/B are important
in maintaining ES cell identity. For this purpose, we extended the comparative
gene expression analysis into sorted genes based on GO term categories. We
found a comparable correlation in genes involved in regulation of
transcription, transcription, development and apoptosis
(Fig. 3B). Notably, the highest
correlation was seen in genes involved in signaling pathways for Notch and
Lif, both of which are implicated in stem cell maintenance
(Androutsellis-Theotokis et al.,
2006; Williams et al.,
1988).
To test whether this observed correlation is statistically significant, we investigated the average expression changes caused by Ring1A/B depletion in genes more than 2-fold derepressed and repressed by Oct3/4 depletion. We further estimated the correlation of the expression changes with the degree of Ring1B binding to the respective genes. On average, derepressed genes in Oct3/4-KO cells were significantly derepressed in the Ring1A/B-dKO, as represented by a value at the zero point on the x-axis (P=1.23x10-39) (Fig. 3C, red circle; see also Table S6 in the supplementary material). The degree of derepression in Ring1A/B-dKO showed a linear correlation with the degree of Ring1B binding (Fig. 3C, red circles). Concordantly, 120 out of 670 genes repressed by Oct3/4 were bound by Ring1B (Fig. 3D). By contrast, repressed genes in the Oct3/4-KO were only slightly repressed in the Ring1A/B-dKO, and these were genes that bound less Ring1B, whereas this was not the case at genes bound by Ring1B at intermediate or high levels (Fig. 3C, blue circles). Taken together with the spontaneous differentiation observed in Ring1A/B-dKO ES cells, Ring1A/B appear to be functionally linked with Oct3/4 in mediating ES cell identity.
Oct3/4 is required to engage PRC1 and PRC2 at target gene promoters
To examine the molecular basis for the functional link between Oct3/4 and
Ring1A/B, we used ChIP to investigate the effect of Oct3/4 deletion on the
levels of Ring1B at selected targets bound by both Ring1B and Oct3/4 and/or
Nanog (Boyer et al., 2005;
Loh et al., 2006). Of the
selected genes Cdx2, Hand1, Gata6 and Hoxb4 were derepressed
1 day after Oct3/4 depletion, whereas T, Otx2 and Hoxb8 were
not (Fig. 4A). Ring1B binding
was significantly reduced irrespective of transcriptional status, suggesting
Oct3/4-mediated regulation Ring1B binding to the chromatin
(Fig. 4B). We extended the
analysis to examine whether this hierarchical link is applicable to other
Ring1B target genes by the ChIP-chip approach. As shown in
Fig. 4C, Ring1B binding to the
promoter regions of the target genes was, on average, significantly reduced 2
days after Tc treatment of ZHBTc4 ES cells. Therefore, binding of Ring1B to
the chromatin in ES cells is generally dependent on Oct3/4. It has been
reported that chromatin binding of Ring1B is also regulated by PRC2 functions
(Boyer et al., 2006). We thus
extended the analysis to address whether Oct3/4-dependent chromatin-binding of
Ring1B involves PRC2, and found that binding of Eed to these genes was
significantly reduced as well (Fig.
4B). Taken together, these results indicate that Oct3/4 mediates
local engagement of PRC1 and PRC2.
|
;
Loh et al., 2006;
Matoba et al., 2006).
Moreover, the co-occupancy of promoters by PRC2 and OCT4/SOX2/NANOG has been
demonstrated only at the transcriptionally repressed genes in human ES cells
(Boyer et al., 2006).
Considered together with the phenotypic difference between Oct3/4-KO
and Ring1A/B-dKO ES cells, the global reduction in PcG binding might
be a part of the mechanism for the differentiation of Oct3/4-KO ES
cells. Finally, we examined whether the binding of Oct3/4 depends on Ring1A/B. The levels of Oct3/4 binding to the PcG target sites were either unchanged or slightly decreased 2 to 4 days after OHT treatment of the Ring1A/B-dKO ES cells (Fig. 4D). Considering that the overall level of Oct3/4 decreases slightly 4 days after OHT treatment (see Fig. S3 in the supplementary material), we conclude that Ring1A/B are not directly required for the binding of Oct3/4 to the target sites.
|
; Niwa et al.,
2005) by maintaining local engagement of PRC1 and PRC2.
Molecular links between Polycomb and the core transcriptional regulatory circuitry
To determine the molecular mechanism for the global reduction of Ring1B
binding upon Oct3/4 depletion, we investigated the effect of Oct3/4 deletion
on the level of PRC1 and PRC2 proteins. Although Ring1B expression was only
minimally affected during the first 48 hours of Tc treatment, expression of
Phc1, Eed and Suz12 was significantly reduced
(Fig. 5A). The decrease in Phc1
and PRC2 proteins was accompanied by a significant reduction in their
respective transcript levels, whereas this was not the case for other PRC1
components, including Ring1B and Bmi1
(Fig. 5B). Therefore, Oct3/4
regulates the expression of PRC1 and PRC2 components, and this may partly
involve transcriptional regulation.
We also investigated whether the physical interaction of Ring1B with the
Rex1 (Zfp42 - Mouse Genome Informatics) complex
(Wang et al., 2006) could be
extended to Oct3/4. Significant amounts of Oct3/4 and Ring1B as well as Rybp,
a Ring1B-binding protein (Garcia et al.,
1999), were found to form complexes in ES cells, whereas the PRC2
protein Suz12 did not co-immunoprecipitate with either Oct3/4 or Ring1B
(Fig. 5C). Since reciprocal
co-immunoprecipitation of Oct3/4 and Ring1B was not affected by the addition
of ethidium bromide, which is known to disrupt protein-DNA interactions
without affecting protein-protein interactions
(Lai and Herr, 1992)
(Fig. 5C, right), this
interaction is not mediated by genomic DNA. This result suggests that the
local binding of PRC1 to chromatin might involve direct interactions between
PRC1 and protein complexes that include Nanog and/or Oct3/4. Taken together,
these results suggest that PRC1 is linked to the core transcriptional
regulatory circuitry at multiple levels.
|
;
Fujikura et al., 2002). We
therefore examined the effects of the enforced expression of Gata6 on PRC1/2
engagement at Ring1B targets using ES cells expressing a Gata6-GR fusion
protein (G6GR) (Shimosato et al.,
2007). As previously described, G6GR cells rapidly lose
ES-cell-like morphology and show dispersed visceral endoderm-like morphology
upon Gata6 activation by the administration of dexamethasone (Dex) (see Fig.
S5 in the supplementary material). We first analyzed the effect of Gata6 activation on the level of PRC1 and PRC2 proteins (Fig. 6A). The level of Ring1B protein was unaffected or only minimally affected during the first 2 days of Dex treatment, but was slightly decreased by day 3. The levels of Phc1, Ezh2, Eed and Suz12 proteins were significantly reduced after Dex treatment, whereas H3K27me3 was unaffected.
Next we used microarray analysis to evaluate the effect of enforced Gata6 expression on gene expression, and compared this profile with those of Oct3/4-KO and Ring1A/B-dKO ES cells. We found a comparable correlation between Gata6-differentiated and Ring1A/B-dKO ES cells (r=0.297), between Oct3/4-KO and Gata6-differentiated ES cells (r=0.318), and between Oct3/4-KO and Ring1A/B-dKO ES cells (r=0.279), not only in terms of total calculable genes but also in genes involved in development, transcription, apoptosis and cell cycle (Fig. 6B). Therefore, a subset of genes induced by enforced Gata6 activation is correlated with those regulated by Oct3/4 and Ring1A/B, suggesting an extension of the functional link to Gata6.
|
|
DISCUSSION |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
The dissociation of PRC1 as a prerequisite for subsequent association of chromatin remodeling components
These and previous studies suggest that PcG proteins are linked to the core
transcriptional regulatory circuitry at multiple levels
(Fig. 3)
(Lee et al., 2006). Oct3/4 is
likely to recruit Ring1B to its targets via direct interactions and also to
induce the expression of PRC1 components via a transcriptional regulatory
mechanism (Fig. 5). Moreover,
it is notable that Oct3/4 loss displaces Ring1B from most of its target genes,
which are not necessarily functional targets of Oct3/4
(Fig. 4C). This prompts us to
postulate an activity that modulates Ring1B recruitment under the regulation
of Oct3/4. Indeed, the RING1 and YY1 binding protein, Rybp, potentially
fulfils such a linking role between Oct3/4 and Ring1B because Rybp is able to
form complexes with both proteins (Fig.
5C) (Wang et al.,
2006). Such multiple interactions might enable coordinated
displacement of PRC1 and PRC2 from their target genes upon disruption of the
core circuitry by differentiation cues. Since forced depletion of
Ring1A/B leads to spontaneous differentiation of ES cells,
dissociation of PRC1 from the targets may be functionally implicated in the
differentiation process. We presume that the dissociation of PRC1 and PRC2 is
a prerequisite for the subsequent association of other chromatin modifiers
such as Trithorax group proteins, which catalyze local hypertrimethylation of
H3K4 upon Oct3/4 depletion (Dou et al.,
2005; Wysocka et al.,
2003). This is supported by our results shown in
Fig. 2C, and by previous
experiments showing that the SWI-SNF complex is unable to remodel
polynucleosomal templates bound by PRC1 in vitro
(Shao et al., 1999).
Therefore, the global enhancement of chromatin remodeling at developmental
genes might be one of the essential events in promoting proper differentiation
of ES cells.
Implications from the reversibility of Polycomb binding in the balance of self-renewal versus differentiation
The reversibility of Polycomb binding to the targets regulated by the core
transcriptional regulatory circuitry and by differentiation cues might confer
self-renewing and differentiation capacities to ES cells. Intriguingly, PcG
silencing has been suggested to be involved in the function and maintenance of
tissue stem and cancer cells, which are also characterized by both
self-renewal and differentiation potency
(Lessard and Sauvageau, 2003;
Molofsky et al., 2003;
Ohta et al., 2002;
Park et al., 2003;
Villa et al., 2007). For
example, Bmi1 loss promotes differentiation of hematopoietic stem
cells (HSCs) and premature senescence of neural stem cells, whereas forced
expression of Bmi1 enhances symmetrical cell division of HSCs
(Iwama et al., 2004;
Molofsky et al., 2005).
Recently, it has been reported that knockdown of SUZ12 in acute
promyelocytic leukemic cells results in myeloid differentiation
(Villa et al., 2007). It is
thus likely that similar molecular mechanisms identified in ES cells that
involve Ring1A/B might operate in the maintenance and differentiation
of various tissue stem cells and cancer cells. Since Oct3/4 and
Nanog are not expressed in most somatic cells, other downstream
effectors expressed in common among the stem cells might be more directly
involved in the regulation of Polycomb binding. Alternatively, other factors
specifically expressed in tissue and/or cancer stem cells might substitute for
the action of Oct3/4 or Nanog. Further studies will be
needed to clarify these issues.
|
|
|
ACKNOWLEDGMENTS |
|---|
|
REFERENCES |
|---|
|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
|---|
Androutsellis-Theotokis, A., Leker, R. R., Soldner, F.,
Hoeppner, D. J., Ravin, R., Poser, S. W., Rueger, M. A., Bae, S. K., Kittappa,
R. and McKay, R. D. (2006). Notch signalling regulates stem
cell numbers in vitro and in vivo. Nature
442,823
-826.[CrossRef][Medline]
Atsuta, T., Fujimura, S., Moriya, H., Vidal, M., Akasaka, T. and
Koseki, H. (2001). Production of monoclonal antibodies
against mammalian Ring1B proteins. Hybridoma
20, 43-46.[CrossRef][Medline]
Auernhammer, C. J. and Melmed, S. (2000).
Leukemia-inhibitory factor-neuroimmune modulator of endocrine function.
Endocr. Rev. 21,313
-345.
Avilion, A. A., Nicolis, S. K., Pevny, L. H., Perez, L., Vivian,
N. and Lovell- Badge, R. (2003). Multipotent cell lineages in
early mouse development depend on SOX2 function. Genes
Dev. 17,126
-140.
Azuara, V., Perry, P., Sauer, S., Spivakov, M., Jorgensen, H.
F., John, R. M., Gouti, M., Casanova, M., Warnes, G., Merkenschlager, M. et
al. (2006). Chromatin signatures of pluripotent cell lines.
Nat. Cell Biol. 8,532
-538.[CrossRef][Medline]
Bernstein, B. E., Mikkelsen, T. S., Xie, X., Kamal, M., Huebert,
D. J., Cuff, J., Fry, B., Meissner, A., Wernig, M., Plath, K. et al.
(2006). A bivalent chromatin structure marks key developmental
genes in embryonic stem cells. Cell
125,315
-326.[CrossRef][Medline]
Bolstad, B. M., Irizarry, R. A., Astrand, M. and Speed, T.
P. (2003). A comparison of normalization methods for high
density oligonucleotide array data based on variance and bias.
Bioinformatics 19,185
-193.
Boyer, L. A., Lee, T. I., Cole, M. F., Johnstone, S. E., Levine,
S. S., Zucker, J. P., Guenther, M. G., Kumar, R. M., Murray, H. L., Jenner, R.
G. et al. (2005). Core transcriptional regulatory circuitry
in human embryonic stem cells. Cell
122,947
-956.[CrossRef][Medline]
Boyer, L. A., Plath, K., Zeitlinger, J., Brambrink, T.,
Medeiros, L. A., Lee, T. I., Levine, S. S., Wernig, M., Tajonar, A., Ray, M.
K. et al. (2006). Polycomb complexes repress developmental
regulators in murine embryonic stem cells. Nature
441,349
-353.[CrossRef][Medline]
Buchwald, G., van der Stoop, P., Weichenrieder, O., Perrakis,
A., van Lohuizen, M. and Sixma, T. K. (2006). Structure and
E3-ligase activity of the Ring-Ring complex of polycomb proteins Bmi1 and
Ring1b. EMBO J. 25,2465
-2474.[CrossRef][Medline]
Burdon, T., Smith, A. and Savatier, P. (2002).
Signalling, cell cycle and pluripotency in embryonic stem cells.
Trends Cell Biol. 12,432
-438.[CrossRef][Medline]
Cales, C., Roman-Trufero, M., Pavon, L., Serrano, I., Melgar,
T., Endoh, M., Perez, C., Koseki, H. and Vidal, M. (2008).
Inactivation of the polycomb group protein Ring1B unveils an antiproliferative
role in hematopoietic cell expansion and cooperation with tumorigenesis
associated with Ink4a deletion. Mol. Cell. Biol.
28,1018
-1028.
Cao, R., Wang, L., Wang, H., Xia, L., Erdjument-Bromage, H.,
Tempst, P., Jones, R. S. and Zhang, Y. (2002). Role of
histone H3 lysine 27 methylation in Polycomb-group silencing.
Science 298,1039
-1043.
Catena, R., Tiveron, C., Ronchi, A., Porta, S., Ferri, A.,
Tatangelo, L., Cavallaro, M., Favaro, R., Ottolenghi, S., Reinbold, R. et
al. (2004). Conserved POU binding DNA sites in the Sox2
upstream enhancer regulate gene expression in embryonic and neural stem cells.
J. Biol. Chem. 279,41846
-41857.
Chambers, I., Colby, D., Robertson, M., Nichols, J., Lee, S.,
Tweedie, S. and Smith, A. (2003). Functional expression
cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.
Cell 113,643
-655.[CrossRef][Medline]
Chazaud, C., Yamanaka, Y., Pawson, T. and Rossant, J.
(2006). Early lineage segregation between epiblast and primitive
endoderm in mouse blastocysts through the Grb2-MAPK pathway. Dev.
Cell 10,615
-624.[CrossRef][Medline]
Czermin, B., Melfi, R., McCabe, D., Seitz, V., Imhof, A. and
Pirrotta, V. (2002). Drosophila enhancer of Zeste/ESC
complexes have a histone H3 methyltransferase activity that marks chromosomal
Polycomb sites. Cell
111,185
-196.[CrossRef][Medline]
Dou, Y., Milne, T. A., Tackett, A. J., Smith, E. R., Fukuda, A.,
Wysocka, J., Allis, C. D., Chait, B. T., Hess, J. L. and Roeder, R. G.
(2005). Physical association and coordinate function of the H3 K4
methyltransferase MLL1 and the H4 K16 acetyltransferase MOF.
Cell 121,873
-885.[CrossRef][Medline]
de Napoles, M., Mermoud, J. E., Wakao, R., Tang, Y. A., Endoh,
M., Appanah, R., Nesterova, T. B., Silva, J., Otte, A. P., Vidal, M. et
al. (2004). Polycomb group proteins Ring1A/B link
ubiquitylation of histone H2A to heritable gene silencing and X inactivation.
Dev. Cell 7,663
-676.[CrossRef][Medline]
del Mar Lorente, M., Marcos-Gutierrez, C., Perez, C.,
Schoorlemmer, J., Ramirez, A., Magin, T. and Vidal, M.
(2000). Loss- and gain-of-function mutations show a polycomb
group function for Ring1A in mice. Development
127,5093
-5100.[Abstract]
Evans, M. J. and Kaufman, M. H. (1981).
Establishment in culture of pluripotential cells from mouse embryos.
Nature 292,154
-156.[CrossRef][Medline]
Fischle, W., Wang, Y., Jacobs, S. A., Kim, Y., Allis, C. D. and
Khorasanizadeh, S. (2003). Molecular basis for the
discrimination of repressive methyl-lysine marks in histone H3 by Polycomb and
HP1 chromodomains. Genes Dev.
17,1870
-1881.
Fujikura, J., Yamato, E., Yonemura, S., Hosoda, K., Masui, S.,
Nakao, K., Miyazaki, J.-i., and Niwa, H. (2002).
Differentiation of embryonic stem cells is induced by GATA factors.
Genes Dev. 16,784
-789.
Fujimura, Y., Isono, K., Vidal, M., Endoh, M., Kajita, H.,
Mizutani-Koseki, Y., Takihara, Y., van Lohuizen, M., Otte, A., Jenuwein, T. et
al. (2006). Distinct roles of Polycomb group gene products in
transcriptionally repressed and active domains of Hoxb8.
Development 133,2371
-2381.
Garcia, E., Marcos-Gutierrez, C., del Mar Lorente, M., Moreno,
J. C. and Vidal, M. (1999). RYBP, a new repressor protein
that interacts with components of the mammalian Polycomb complex, and with the
transcription factor YY1. EMBO J.
18,3404
-3418.[CrossRef][Medline]
Hamer, K. M., Sewalt, R. G., den Blaauwen, J. L., Hendrix, T.,
Satijn, D. P. and Otte, A. P. (2002). A panel of monoclonal
antibodies against human polycomb group proteins. Hybrid
Hybridomics 21,245
-252.[CrossRef][Medline]
Heinrich, P. C., Behrmann, I., Haan, S., Hermanns, H. M.,
Muller-Newen, G. and Schaper, F. (2003). Principles of
interleukin (IL)-6-type cytokine signalling and its regulation.
Biochem. J. 374,1
-20.[CrossRef][Medline]
Isono, K., Fujimura, Y.-i, Shinga, J., Yamaki, M., O-Wang, J.,
Takihara, Y., Murahashi, Y., Takada, Y., Mizutani-Koseki, Y. and Koseki,
H. (2005a). Mammalian polyhomeotic homologues Phc2 and Phc1
act in synergy to mediate polycomb repression of Hox genes. Mol.
Cell. Biol. 25,6694
-6706.
Isono, K., Mizutani-Koseki, Y., Komori, T., Schmidt-Zachmann, M.
S. and Koseki, H. (2005b). Mammalian polycomb-mediated
repression of Hox genes requires the essential spliceosomal protein Sf3b1.
Genes Dev. 19,536
-541.
Iwama, A., Oguro, H., Negishi, M., Kato, Y., Morita, Y., Tsukui,
H., Ema, H., Kamijo, T., Katoh-Fukui, Y., Koseki, H. et al.
(2004). Enhanced self-renewal of hematopoietic stem cells
mediated by the polycomb gene product Bmi-1. Immunity
21,843
-851.[CrossRef][Medline]
Kuroda, T., Tada, M., Kubota, H., Kimura, H., Hatano, S. Y.,
Suemori, H., Nakatsuji, N. and Tada, T. (2005). Octamer and
Sox elements are required for transcriptional cis regulation of Nanog gene
expression. Mol. Cell. Biol.
25,2475
-2485.
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.
Lai, J. S. and Herr, W. (1992). Ethidium
bromide provides a simple tool for identifying genuine DNA-independent protein
associations. Proc. Natl. Acad. Sci. USA
89,6958
-6962.
Lee, T. I., Jenner, R. G., Boyer, L. A., Guenther, M. G.,
Levine, S. S., Kumar, R. M., Chevalier, B., Johnstone, S. E., Cole, M. F.,
Isono, K. et al. (2006). Control of developmental regulators
by Polycomb in human embryonic stem cells. Cell
125,301
-313.[CrossRef][Medline]
Leeb, M. and Wutz, A. (2007). Ring1B is crucial
for the regulation of developmental control genes and PRC1 proteins but not X
inactivation in embryonic cells. J. Cell Biol.
178,219
-229.
Lei, H., Oh, S. P., Okano, M., Juttermann, R., Goss, K. A.,
Jaenisch, R. and Li, E. (1996). De novo DNA cytosine
methyltransferase activities in mouse embryonic stem cells.
Development 122,3195
-3205.[Abstract]
Lessard, J. and Sauvageau, G. (2003). Bmi-1
determines the proliferative capacity of normal and leukaemic stem cells.
Nature 423,255
-260.[CrossRef][Medline]
Loh, Y. H., Wu, Q., Chew, J. L., Vega, V. B., Zhang, W., Chen,
X., Bourque, G., George, J., Leong, B., Liu, J. et al.
(2006). The Oct4 and Nanog transcription network regulates
pluripotency in mouse embryonic stem cells. Nat.
Genet. 38,431
-440.[CrossRef][Medline]
Martin, G. R. (1981). Isolation of a
pluripotent cell line from early mouse embryos cultured in medium conditioned
by teratocarcinoma stem cells. Proc. Natl. Acad. Sci.
USA 78,7634
-7638.
Matoba, R., Niwa, H., Masui, S., Ohtsuka, S., Carter, M. G.,
Sharov, A. A. and Ko, M. S. (2006). Dissecting
oct3/4-regulated gene networks in embryonic stem cells by expression
profiling. PLoS ONE 1,e26
.[CrossRef]
Min, J., Zhang, Y. and Xu, R. M. (2003).
Structural basis for specific binding of Polycomb chromodomain to histone H3
methylated at Lys 27. Genes Dev.
17,1823
-1828.
Mitsui, K., Tokuzawa, Y., Itoh, H., Segawa, K., Murakami, M.,
Takahashi, K., Maruyama, M., Maeda, M. and Yamanaka, S.
(2003). The homeoprotein Nanog is required for maintenance of
pluripotency in mouse epiblast and ES cells. Cell
113,631
-642.[CrossRef][Medline]
Miyagishima, H., Isono, K., Fujimura, Y., Iyo, M., Takihara, Y.,
Masumoto, H., Vidal, M. and Koseki, H. (2003). Dissociation
of mammalian Polycomb-group proteins, Ring1B and Rae28/Ph1, from the chromatin
correlates with configuration changes of the chromatin in mitotic and meiotic
prophase. Histochem. Cell Biol.
120,111
-119.[CrossRef][Medline]
Molofsky, A. V., Pardal, R., Iwashita, T., Park, I. K., Clarke,
M. F. and Morrison, S. J. (2003). Bmi-1 dependence
distinguishes neural stem cell self-renewal from progenitor proliferation.
Nature 425,962
-967.[CrossRef][Medline]
Molofsky, A. V., He, S., Bydon, M., Morrison, S. J. and Pardal,
R. (2005). Bmi-1 promotes neural stem cell self-renewal and
neural development but not mouse growth and survival by repressing the
p16Ink4a and p19Arf senescence pathways. Genes Dev.
19,1432
-1437.
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.[CrossRef][Medline]
Nichols, J., Zevnik, B., Anastassiadis, K., Niwa, H.,
Klewe-Nebenius, D., Chambers, I., Scholer, H. and Smith, A.
(1998). Formation of pluripotent stem cells in the mammalian
embryo depends on the POU transcription factor Oct4.
Cell 95,379
-391.[CrossRef][Medline]
Niwa, H., Miyazaki, J. and Smith, A. G. (2000).
Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation
or self-renewal of ES cells. Nat. Genet.
24,372
-376.[CrossRef][Medline]
Niwa, H., Toyooka, Y., Shimosato, D., Strumpf, D., Takahashi,
K., Yagi, R. and Rossant, J. (2005). Interaction between
Oct3/4 and Cdx2 determines trophectoderm differentiation.
Cell 123,917
-929.[CrossRef][Medline]
Ohta, H., Sawada, A., Kim, J. Y., Tokimasa, S., Nishiguchi, S.,
Humphries, R. K., Hara, J. and Takihara, Y. (2002). Polycomb
group gene rae28 is required for sustaining activity of hematopoietic stem
cells. J. Exp. Med. 195,759
-770.
Okumura-Nakanishi, S., Saito, M., Niwa, H. and Ishikawa, F.
(2005). Oct-3/4 and Sox2 regulate Oct-3/4 gene in embryonic stem
cells. J. Biol. Chem.
280,5307
-5317.
Orlando, V., Strutt, H. and Paro, R. (1997).
Analysis of chromatin structure by in vivo formaldehyde cross-linking.
Methods 11,205
-214.[CrossRef][Medline]
Park, I. K., Qian, D., Kiel, M., Becker, M. W., Pihalja, M.,
Weissman, I. L., Morrison, S. J. and Clarke, M. F. (2003).
Bmi-1 is required for maintenance of adult self-renewing haematopoietic stem
cells. Nature 423,302
-305.[CrossRef][Medline]
Rodda, D. J., Chew, J. L., Lim, L. H., Loh, Y. H., Wang, B., Ng,
H. H. and Robson, P. (2005). Transcriptional regulation of
nanog by OCT4 and SOX2. J. Biol. Chem.
280,24731
-24737.
Seibler, J., Zevnik, B., Kuter-Luks, B., Andreas, S., Kern, H.,
Hennek, T., Rode, A., Heimann, C., Faust, N., Kauselmann, G. et al.
(2003). Rapid generation of inducible mouse mutants.
Nucleic Acids Res. 31,e12
.
Shao, Z., Raible, F., Mollaaghababa, R., Guyon, J. R., Wu, C.
T., Bender, W. and Kingston, R. E. (1999). Stabilization of
chromatin structure by PRC1, a Polycomb complex. Cell
98, 37-46.[CrossRef][Medline]
Shimosato, D., Shiki, M. and Niwa, H. (2007).
Extra-embryonic endoderm cells derived from ES cells induced by GATA Factors
acquire the character of XEN cells. BMC Dev Biol.
7, 80.[CrossRef][Medline]
Villa, R., Pasini, D., Gutierrez, A., Morey, L., Occhionorelli,
M., Vire, E., Nomdedeu, J. F., Jenuwein, T., Pelicci, P. G., Minucci, S. et
al. (2007). Role of the polycomb repressive complex 2 in
acute promyelocytic leukemia. Cancer Cell
11,513
-525.[CrossRef][Medline]
Wang, H., Wang, L., Erdjument-Bromage, H., Vidal, M., Tempst,
P., Jones, R. S. and Zhang, Y. (2004). Role of histone H2A
ubiquitination in Polycomb silencing. Nature
431,873
-878.[CrossRef][Medline]
Wang, J., Rao, S., Chu, J., Shen, X., Levasseur, D. N.,
Theunissen, T. W. and Orkin, S. H. (2006). A protein
interaction network for pluripotency of embryonic stem cells.
Nature 444,364
-368.[CrossRef][Medline]
Williams, R. L., Hilton, D. J., Pease, S., Willson, T. A.,
Stewart, C. L., Gearing, D. P., Wagner, E. F., Metcalf, D., Nicola, N. A. and
Gough, N. M. (1988). Myeloid leukaemia inhibitory factor
maintains the developmental potential of embryonic stem cells.
Nature 336,684
-687.[CrossRef][Medline]
Wysocka, J., Myers, M. P., Laherty, C. D., Eisenman, R. N. and
Herr, W. (2003). Human Sin3 deacetylase and trithorax-related
Set1/Ash2 histone H3-K4 methyltransferase are tethered together selectively by
the cell-proliferation factor HCF-1. Genes Dev.
17,896
-911.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati 
Twitter What's this?
Related articles in Development:
This article has been cited by other articles:
|
|
 |
|
  P. Yuan, J. Han, G. Guo, Y. L. Orlov, M. Huss, Y.-H. Loh, L.-P. Yaw, P. Robson, B. Lim, and H.-H. Ng Eset partners with Oct4 to restrict extraembryonic trophoblast lineage potential in embryonic stem cells Genes & Dev., November 1, 2009; 23(21): 2507 - 2520. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 B. Jin, B. Yao, J.-L. Li, C. R. Fields, A. L. Delmas, C. Liu, and K. D. Robertson DNMT1 and DNMT3B Modulate Distinct Polycomb-Mediated Histone Modifications in Colon Cancer Cancer Res., September 15, 2009; 69(18): 7412 - 7421. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Pasini, A.P. Bracken, K. Agger, J. Christensen, K. Hansen, P.A.C. Cloos, and K. Helin Regulation of Stem Cell Differentiation by Histone Methyltransferases and Demethylases Cold Spring Harb Symp Quant Biol, November 6, 2008; (2008) sqb.2008.73.009v1. [Abstract] [PDF]
|
|
|
|
|
|
M.F. Cole and R.A. Young Mapping Key Features of Transcriptional Regulatory Circuitry in Embryonic Stem Cells Cold Spring Harb Symp Quant Biol, November 6, 2008; (2008) sqb.2008.73.027v1. [Abstract] [PDF]
|
|
|
|
 |
|
 M. Endoh, T. A. Endo, T. Endoh, Y.-i. Fujimura, O. Ohara, T. Toyoda, A. P. Otte, M. Okano, N. Brockdorff, M. Vidal, et al. Polycomb group proteins Ring1A/B are functionally linked to the core transcriptional regulatory circuitry to maintain ES cell identity J. Cell Sci., April 15, 2008; 121(8): e806 - e806. [Full Text]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
