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First published online 10 May 2006
doi: 10.1242/dev.02405
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1 RIKEN Research Center for Allergy and Immunology, 1-7-22 Suehiro, Tsurumi-ku,
Yokohama 230-0045, Japan.
2 Centro de Investigaciones Biologicas, Department of Developmental and Cell
Biology, Ramiro de Maeztu 9, 28040 Madrid, Spain.
3 Department of Stem Cell Biology, Research Institute for Radiation Biology and
Medicine, Hiroshima University, 1-2-3 Kasumi, Minami-ku, Hiroshima 734-8553,
Japan.
4 Division of Molecular Genetics, The Netherlands Cancer Institute, 1066CX
Amsterdam, The Netherlands.
5 Swammerdam Institute for Life Sciences, University of Amsterdam, Kruislaan
406, 1098 SM Amsterdam, The Netherlands.
6 Research Institute of Molecular Pathology, The Vienna Biocenter, Dr Bohrgasse
7, A-1030 Vienna, Austria.
7 Hubrecht Laboratory, Uppsalalaan 8 3584CT Utrecht, The Netherlands.
Author for correspondence (e-mail:
fujimara{at}rcai.riken.jp)
Accepted 12 April 2006
 |
SUMMARY |
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 TOP SUMMARY INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key words: Polycomb, Hox, Mouse, Chromatin, Immunoprecipitation
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INTRODUCTION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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;
Pirrotta, 1997). Indeed, some
of the PcG genes have been identified as suppressors of trxG mutations
(Kennison and Tamkun, 1988).
The trxG and PcG genes encode nuclear factors that, by
forming multimeric protein complexes on the chromatin, freeze transcriptional
states determined early in embryogenesis. A major function of trxG gene
products appears to concern the remodeling of chromatin structure, as several
of these genes encode subunits of the SWI/SNF chromatin remodeling complex and
associate with catalytic activities that modify core histone tails
(Milne et al., 2002;
Nakamura et al., 2002;
Tamkun et al., 1992). PcG gene
products have been shown to form at least two types of multimeric protein
complexes (Shao et al., 1999;
Czermin et al., 2002;
Muller et al., 2002;
Cao et al., 2002). The first
type of complex, known as class 1, includes Extra sex combs (Esc) and Enhancer
of zeste [E(z)], and its association with histone deacetylase and
methyltransferase activities suggests a function in the modification of
histone tails (Czermin et al.,
2002; Muller et al.,
2002; Cao et al.,
2002). The second type of complex (class 2) contains Polycomb
(Pc), Posterior sex combs (Psc), Polyhomeotic (Ph), Dring (Sex comb extra;
Sce) and Sex comb on midleg (Scm) in Drosophila
(Shao et al., 1999). It has
been shown that, by interacting with DNA regulatory sequences termed PcG
response elements (PREs), this complex can maintain the transcriptional
silence of target genes. Recently, it has been reported that class 1
PcG-mediated trimethylation of histone H3 lysine 27 (H3-K27) serves as a
signal for the recruitment of class 2 PcG complexes, which in turn may silence
target genes flanked by PREs (Cavalli and
Paro, 1998; Wang et al.,
2004b). Purified class 2 core complexes efficiently block
SWI/SNF-dependent remodeling of nucleosomal arrays and subsequent
transcription in vitro (King et al.,
2002; Levine et al.,
2002; Shao et al.,
1999).
In mammals, it has been demonstrated that the expression of Hox cluster
genes depends on the control of mammalian homologues of trxG and both classes
of PcG gene products. Silencing of several Hox gene expressions has been
observed in mice lacking Mll, a mammalian homologue of Drosophila
trx, between 8.5 and 9.5 days post coitus (dpc)
(Yu et al., 1998). Conversely,
a hypomorphic mutation of Eed, a homologue of Drosophila
Esc, revealed its involvement in repressing Hox gene expression
(Schumacher et al., 1996). As Mll and class 1 PcG complex are known to be
associated with activities that modify histone tails
(van der Vlag and Otte, 1999),
such modifications could be involved in maintaining spatially restricted
expression of Hox cluster genes. Indeed, it has been shown that the Mll
protein regulates the acetylation of lysine 4 of histone H3 (H3-K4) at several
Hox genes, whereas Ezh2, a homologue of E(z), mediates H3-K27 trimethylation
(Milne et al., 2002;
Nakamura et al., 2002). The
involvement of class 2 PcG in this maintenance has been revealed previously by
the effect of mutations in Bmi1 (Pcgf4 - Mouse Genome
Informatics), Rnf110 (Mel18; Pcgf2 - Mouse Genome
Informatics), Phc1 (rae28) and Rnf2
(Ring1B) on Hox gene expression
(Akasaka et al., 1996;
van der Lugt et al., 1996;
Suzuki et al., 2002;
Takihara et al., 1997).
Notably, in mice doubly deficient for Rnf110 and Bmi1,
homologues of Drosophila Psc, Hoxb6 expression was normally localized
in caudal tissues of 8.5 dpc embryos, but was progressively de-repressed
cranially thereafter (Akasaka et al.,
2001). Thus, class 2 PcG proteins may participate in maintaining
transcriptionally silent states of Hox genes outside their expression domains.
However, to date, the molecular mechanisms used by mammalian trxG and PcG gene
products to regulate Hox genes is not well understood, in part because the
relationship between PcG protein binding and histone tail modifications has
not yet been widely documented around the Hox loci in developing embryos. We
have addressed this issue by documenting the association of PcG proteins,
H3-K9 acetylation, H3-K4 methylation (marks of transcriptionally active
chromatin) and H3-K27 trimethylation (a mark of transcriptionally inactive
chromatin) to the genomic region flanking Hoxb8, a Hox gene known to
require the class 2 PcG proteins for its posterior restriction.
We show that the association of PcG proteins, H3-K9 acetylation, H3-K4 methylation and H3-K27 trimethylation around Hoxb8 differs in embryonic tissues expressing and not expressing the gene. By using mutant alleles for Rnf2, which encode constituents of class 2 PcG complexes, and Suz12 causing a strong decrease in H3-K27 methylation, we show that the recruitment of the class 2 PcG complex, mediated by trimethylated H3-K27, plays a decisive role in maintaining the repression of Hox genes outside their expression domain, as it is the case in Drosophila. The positive role of class 2 PcG complex proteins in the transcriptionally active domain was shown to involve the regulation of H3-K9 acetylation.
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MATERIALS AND METHODS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). The generation of Suz12-deficient mice and ES
cells will be described in a separate publication. CRE-mediated deletion of
Rnf2 in MEF cell lines using adenovirus expressing the CRE recombinase (AdCre)
was carried out as described (Kanegae et
al., 1995). AdCre virus was concentrated to be
2.0x10-9 i.f.u./ml and mouse embryonic fibroblast (MEFs) were
infected with AdCre virus at MOI5.
Antibodies
Antibodies used are listed in the Table
1.
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). This
process was bypassed in some cases. Correct amounts of NaCl and NP-40 were
added to the chromatin fraction or whole cell lysates (WCE) in order to
perform optimal immunoprecipitation for each antibody. Precleared protein
extracts were incubated with the correct amount of antibodies, at 4°C with
rocking, for 2 hours to overnight. Immune complexes were captured after 3
hours incubation with Protein A Sepharose beads. To isolate genomic DNA from immune complexes, beads were treated with 50 µg/ml of RNaseA at 37°C for 30 minutes followed by overnight incubation with 500 µg/ml proteinase K/0.5% SDS at 37°C. After 3 hours heating at 65°C for reverse crosslinking, supernatants were collected, extracted by phenol-chloroform and concentrated by ethanol precipitation. Genomic DNA was also isolated from the original chromatin fraction or WCE through the same procedure as described above and designated as `Input' DNA (see Figs 1, 2, 3). To measure the DNA yield after immunoprecipitation, the aliquots of immunoprecipitated DNA were electrophoresed for 5 minutes in an agarose gel, next to serially diluted input DNA and band-intensities were compared after ethidium bromide staining (see Fig. S2A in the supplementary material).
Equivalent amounts of immunoprecipitated DNA to that of `Input' DNA loaded
in lane `1' were subjected to PCR reactions. Usually, 10 to 20 ng of genomic
DNA was used. Mock-immunoprecipitated DNA (A- and P-) derived from the same
volume of the chromatin fraction as used for anti-Rnf2 immunoprecipitation
were subjected to the PCR. To carry out semi-quantitative PCR, serially
diluted `Input' DNA and immunoprecipitated DNA were used as templates. The
relative quantity of each genomic region in immunoprecipitated genomic DNA was
estimated by referring to the serial dilutions of `Input' DNA isolated from
the initial lysates and an enrichment value was determined. Every series of
experiments were performed at least three times (see Fig. S3 in the
supplementary material). Primers used in this study are listed in
Table 2. ChIP analysis by using
ES cells was performed as described (Isono
et al., 2005a).
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). Quantity of total cellular RNA subjected to
reverse transcriptase (RT)-PCR for Hox genes was adjusted by the expression of
ß-actin. Hoxb8 expression shown in
Fig. 3A was quantified by
referring to Gapdh expression by real-time PCR analyses using Mx3005P
multiplex quantitative PCR systems (Stratagene). Preparation of whole-cell
extracts from embryos, ES cells and MEFs and western blot analysis were
performed as described previously (Isono
et al., 2005a). |
RESULTS |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Quantity of immunoprecipitated genomic DNA was measured by referring to
serially diluted genomic DNA isolated from the initial lysates (see Fig. S2A
in the supplementary material). Usually, for a given volume of chromatin
fraction, the amount of DNA immunoprecipitated by anti-Rnf2 antibodies was
four- to eightfold higher than that brought down in the absence of primary
antibody (see Fig. S2A in the supplementary material). Amounts of DNA from
respective tissues were further adjusted by PCR for the tbx2 gene,
which is also bound by Rnf2 in embryonic tissues (see Fig. S2B in the
supplementary material). Equivalent amounts of DNA immunoprecipitated from the
anterior and posterior tissues were subjected to semi-quantitative PCR using
pairs of primers defining discrete regions in the Hoxb locus
(Fig. 1F, top; see
Table 2). Serially diluted
genomic DNA isolated from the initial lysates was also subjected to the PCR to
evaluate the enrichment value for each region of Hoxb8 in
immunoprecipitated materials (Fig.
1B-E). The adam34 gene, which is expressed exclusively in
adult testes, is not bound by any of Rnf2, Ring1, Phc1, Cbx2 or Rnf110 in
embryonic tissues and thus turned out to serve as a negative control
(Brachvogel et al., 2002)
(Fig. 1B-E).
The association of Rnf2 with regions 1, 2, 3, 9, 10, 11, 13, 15, 16 and D1
was more than four times different in cranial and caudal tissues, whereas this
was not the case for regions 4, 5, 6, 7, 8, 12, 14, 19, 20, 21, 22, 23, 24, 25
or D2 (Fig. 1B,F, Supplemental
Fig. 3). In region 17 and 18, no significant association of Rnf2 was observed.
Regions 1 and 2, and 10 and 11, correspond to the Hoxb8 regulatory
regions BH1100 and KA that have previously been identified by transgenic
approaches; regions 3 and 13 include the promoters of Hoxb8 and
Hoxb7, respectively
(Charité et al., 1995;
Vogels et al., 1993).
Therefore, Rnf2 differentially binds to the proximal cis-regulatory elements
of 5' Hoxb genes in cranial and caudal embryonic tissues.
Likewise, a significant differential association of Rnf2 to the distal element
(DE) located between Hoxb4 and Hoxb5
(Oosterveen et al., 2003) was
also seen in cranial and caudal tissues. Similar to Rnf2, Ring1 association
was seen in cranial tissues, except for regions 3 and 7
(Fig. 1D). In contrast to Rnf2
binding, the association of Phc1 extended through all regions examined except
for region 8, without any obvious differences between cranial and caudal
tissues (Fig. 1C).
Nevertheless, in region 3, the association did appear to be significantly
stronger in the caudal tissues. Furthermore, there was no significant
difference between cranial and caudal tissues with respect to the chromatin
association of Cbx2 (Fig. 1D).
Therefore, these results indicate that Phc1 and Cbx2 are bound to
Hoxb8 genomic region irrespective of the transcriptional state of the
gene. Rnf110 association to regions 3 and 10 was seen in the cranial tissues
only, but to regions 1, 2, 7 and 14 it was seen in both cranial and caudal
tissues (Fig. 1E). These
experiments were performed three times with similar results (see Fig. S3 in
the supplementary material). In summary, the complete form of the class 2 PcG
complexes predominantly associate with Hoxb8 in tissues where the
gene is repressed, whereas form(s) lacking at least the Rnf2 component also
bind in tissues actively expressing the Hox gene.
|
). A
linear correlation between histone modifications and the transcriptional state
of Hox genes has been seen in developing embryos at Hoxd4 locus and
MEFs that are derived from Mll mutants and wild-type controls
(Rastegar et al., 2004;
Milne et al., 2002).
Therefore, as H3-K9 acetylation and H3-K4 methylation may be prerequisites for
active transcription of Hox genes, these extents were compared between
anterior and posterior embryonic tissues. H3-K9 acetylation and H3-K4
methylation around the Hoxb8 was more abundant in the
transcriptionally active caudal embryonic part than in the non-expressing
anterior part as reported by Rastegar et al.
(Rastegar et al., 2004)
(Fig. 2A,B).
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; Lachner et al.,
2003). In particular, trimethylation of H3-K27 has been shown to
be mediated by class 1 PcG complexes (Cao
et al., 2002). The degree of H3-K9 and H3-K27 methylation at
regions 2, 3 and 8 was compared between cranial and caudal embryonic regions.
At all the regions examined, H3-K27 trimethylation was more abundant in the
transcriptionally silent cranial embryonic part than in the Hox-expressing
caudal part (Fig. 2B). No
significant levels of di- or trimethylation of H3-K9 or dimethylation of
H3-K27 were seen at any of the regions examined (Y.F. and H.K.,
unpublished).
Developmental kinetics of Rnf2-association and of histone H3 modifications at Hoxb8
Loss of spatial restriction of Hoxb6 expression, between 8.5 and
9.5 dpc, has been reported as resulting from the Rnf110/Bmi1
double mutation (Akasaka et al.,
2001). Similarly, Mll deficiency progressively silences
Hox gene expression within this time window
(Yu et al., 1998). This
suggests that this developmental stage might be the crucial period when PcG
association and H3-K9 acetylation at Hoxb8 play their role in
modulating gene expression.
Prior to the ChIP analyses, we re-examined the expression of Hoxb8
gene in the cranial and caudal tissues in quantitative manner by using
real-time PCR (Fig. 3A). As
reported previously by extensive in situ hybridization analyses, caudally
restricted expression of Hoxb8 was already established at 8.0 dpc and
its relative quantity progressively increased until 12.5 dpc
(Deschamps and Wijgerde,
1993). We carried out the kinetic analyses of the Rnf2
association, H3-K9 acetylation and H3-K27 trimethylation in early to later
developmental stages across the Hoxb8 promoter region (region 3 in
Fig. 1F). In 8.0 dpc embryos,
no Rnf2 association was seen at the six-somite stage. It was first seen,
exclusively in cranial tissue, at the eight-somite stage
(Fig. 3B). This was also the
case at later stages up to 12.5 dpc (Fig.
3C-E). The relative quantity of Rnf2 association gradually
increased and reached a maximal level at 10.5 dpc
(Fig. 3E). H3-K9 acetylation
was observed in the cranial region at the six-somite stage and in both cranial
and caudal regions at the eight-somite stage. It was present at higher levels
in the caudal region in 9.5 and 10.5 dpc embryos than in 8.0 dpc. H3-K27
trimethylation at region 3 continued to be seen in the cranial, but not in the
caudal, region from 8.5 to 12.5 dpc. In summary, a differential association of
Rnf2 with the Hoxb8 promoter region, region 3, was established from
8.0 dpc onwards, and reached completion around 10.5 dpc
(Fig. 3G)
(Deschamps and Wijgerde,
1993). Relative amounts of acetylated H3-K9 in this region also
increased up to 12.5 dpc. Likewise, differential trimethylation of H3-K27 was
already established at 9.5 dpc and maintained to 12.5 dpc. These observations
indicate that the Rnf2 association, H3-K9 acetylation and H3-K27
trimethylation may be involved to maintain the spatially restricted expression
of the Hoxb8.
|
).
However, it was not determined whether Rnf2 association was functionally
involved in the maintenance rather than early establishment of this repressed
status. To address this question, we conditionally depleted functional
Rnf2 after the expression domain of Hoxb8 was established.
Because of early embryonic lethality of Rnf2-null mice, we generated
a conditional allele designated as Rnf2fl in which exon 2,
containing the ATG initiation codon, was flanked with loxP sites
(Voncken et al., 2003;
de Napoles et al., 2004).
Primary MEFs were derived from the cranial part of
Rnf2fl/fl 9.5 dpc embryos in which Hoxb8
expression was repressed. Subsequent deletion of Rnf2 was achieved by
infection of MEF cultures with adenovirus expressing the CRE recombinase
(Ad-Cre) as described (de Napoles et al.,
2004). Rnf2 depletion was evident in western analysis of infected
cell culture (Fig. 4A) and took
2 days for completion. The analysis of Hoxb8 expression continued for
4 days after Ad-Cre infection. Two independent experiments demonstrated more
than a 16-fold increase of Hoxb8 expression in infected MEFs compared
with the uninfected control. Therefore Rnf2 association is essential for the
maintenance of the transcriptional repression of Hoxb8.
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; Chambeyron et al.,
2005).
Seven Rnf2fl/fl ES cells have been derived
(de Napoles et al., 2004).
Rnf2-/- ES cell derivatives of one of these lines, the
male ES cell line, 13-3, were generated by CRE-mediated excision of exon 2
(Fig. 4B). Loss of functional
Rnf2 resulted in de-repression of Hoxa1, Hoxa7, Hoxb3, Hoxb8, Hoxb13,
Hoxc9, Hoxd8 and Hoxd10 but not of Hoxc8
(Fig. 4C). To test if
de-repression of Hox genes is a direct consequence of mutating Rnf2,
complementation experiments were carried out, transfecting mutant ES cells
with a construct expressing Myc-tagged Rnf2. As shown in
Fig. 4B, transfected mutant ES
cells expressed transgene-encoded Rnf2 at a level equivalent to half that of
endogenous Rnf2. RT-PCR analysis revealed that the expression of Hoxa1,
Hoxb3, Hoxb8 and Hoxd8 was repressed albeit not to the same
degree as in the parental Rnf2fl/fl ES cells
(Fig. 4C). These results
confirm that Rnf2 association is required to mediate the transcriptional
repression of Hox genes in ES cells.
Next, the method by which Rnf2 deficiency impacts on the functions of class
2 PcG complexes was investigated. As it has been suggested that Ring1 is an
important component in the stabilization of the Polycomb core complex in Sf9
cells (Francis et al., 2001),
the expression levels of other components of class 2 PcG complexes in
Rnf2-/- ES cells was examined. The expression of both Phc1
and Cbx2 gene products was obviously reduced in Rnf2-/- ES
cells (Fig. 4D; M.E. and H.K.,
unpublished). By contrast, the expression of RYBP, another Rnf2-binding
protein, which is not found in hPRC-H complex, was not altered
(Garcia et al., 1999)
(Fig. 4D). As the transcription
of Phc1 and Cbx2 was not altered in
Rnf2-/- ES cells, Rnf2 loss specifically affects the
expression of Phc1 and Cbx2. It is thus likely that Rnf2 impacts the Hox
expression by regulating the stability of the class 2 PcG complexes.
The coincidence of Rnf2 association and H3-K27 trimethylation in transcriptionally repressed region further prompted us to ask whether de-repression of Hox genes in Rnf2-/- ES cells involves a change of H3-K27 trimethylation across the Hox genomic regions. The expression of Suz12 and Ezh2 were not significantly changed in Rnf2-/- ES cells (Fig. 4D). Concordant with this result, ChIP analyses revealed that local level of H3-K27 trimethylation at Hox promoter regions were almost unchanged (Fig. 4E). Therefore, Rnf2 deficiency affects Hox gene expressions without changing local H3-K27 trimethylation.
Functional involvement of trimethylated H3-K27 on Rnf2 association at Hox loci and on Hox gene expression
In Drosophila, trimethylation on H3-K27 has been shown to
facilitate the recruitment of class 2 PcG complexes via direct interaction
between trimethylated H3-K27 and the chromodomain of Pc
(Cao et al., 2002;
Czermin et al., 2002;
Muller et al., 2002).
Therefore, Hox gene expression and Rnf2 association at the Hoxb
locus, in the absence of trimethylated H3-K27, was examined using
Suz12-/- ES cells. We have independently generated a
loss-of-function allele of Suz12 and the homozygous mutants exhibited
a phenotype almost identical to that reported by Pasini et al.
(Pasini et al., 2004) (K.I.
and H.K., unpublished). Suz12-/- ES cells were derived
from crosses of heterozygous mutants. The absence of Suz12 reduced the levels
of H3-K27 tri- and dimethylation to less than 10% of the wild type but did not
significantly change H3-K9 methylation
(Pasini et al., 2004). First,
the association of class 1 PcG and H3-K27 trimethylation at the Hox promotor
regions was investigated. Anti-Suz12, -Eed and -trimethylated H3-K27
antibodies specifically immunoprecipitated significant amounts of Hox promoter
fragments from the wild-type ES cells (Fig.
5A). In Suz12-/- ES cells, no Hox DNA could be
detected at all upon anti-Suz12 immunoprecipitation. The loss of Suz12
significantly reduced Eed association and H3-K27 trimethylation at Hox genes.
Therefore, class 1 PcG complexes associate locally to Hox genes and mediate
local H3-K27 trimethylation in undifferentiated ES cells. Second, the
expression of Hoxa1, Hoxa4, Hoxb1, Hoxb3, Hoxb4, Hoxb6, Hoxb8, Hoxb9
and Hoxc6 was compared between wild-type and
Suz12-/- ES cells by RT-PCR. Suz12-/-
ES cells were shown to express more Hox gene transcripts than the wild type
(Fig. 5B). Therefore, local
H3-K27 trimethylation mediated by class 1 PcG complexes, or association of the
complexes, are likely to be required in order to mediate repression of Hox
genes. We went on to examine Rnf2 association in Suz12-/-
ES cells. Rnf2 association to the Hox promoters was significantly reduced in
Suz12-/- ES cells (Fig.
5A). Therefore H3-K27 trimethylation may be a prerequisite for the
association of Rnf2 with Hox regulatory regions, as already demonstrated in
Drosophila. The role of class 1 PcG complexes in the recruitment of
the class 2 complexes via protein-protein interactions is not necessarily
excluded.
|
). Null mutations in the Rnf110 and
Phc1 loci have been shown to decrease the transcription level of
endogenous Hoxb1, and to severely impair the transcription from
Hox/lacZ reporters and knock-in loci
(de Graaff et al., 2003). In
addition, a positive action of PcG complexes explains the drop in gene
expression levels within the Hoxb8 expression domains known to occur
around 9.5 dpc in Bmi1/Rnf110 and Phc1/Phc2 double
mutants (Akasaka et al., 2001;
de Graaff et al., 2003;
Isono et al., 2005b). As the
level of histone H3 acetylation correlates with Hox gene expression in
Mll mutants and controls, and Mll and Bmi1 proteins display discrete
subnuclear colocalization, we investigated whether the drop in Hoxb8
expression level in Rnf110/Bmi1 and
Phc1/Phc2 double mutants involved a change in H3-K9
acetylation (Hanson et al.,
1999; Milne et al.,
2002; de Graaff et al.,
2003). The degree of H3-K9 acetylation in the caudal region of
Rnf110-/-Bmi1-/- embryos was much
lower than in Rnf110+/+Bmi1+/-,
whereas the H3-K9 acetylation at the ß-actin promoter was almost
equivalent (Fig. 6A).
Similarly, H3-K9 acetylation level in the caudal part of
Phc1-/-Phc2-/- was equivalent to the
cranial part whereas craniocaudally graded acetylation was seen in
Phc1+/-Phc2+/- embryos
(Fig. 6B). Therefore
association of class 2 PcG gene products to Hoxb8 is required for the
maintenance of H3-K9 acetylation in transcriptionally active regions.
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|
DISCUSSION |
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TOPSUMMARYINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Rnf2 association to known regulatory elements of the Hoxb8 gene is
seen predominantly in transcriptionally silent anterior embryonic tissues,
whereas the binding of other PcG class 2 members, Phc1 and Cbx2, is observed
at all AP levels, irrespective of transcriptional status. This implies that
different forms of class 2 PcG complexes bind to the Hoxb genomic region in
embryonic domains where the gene is transcriptionally active and repressed.
This is reminiscent of previous findings in the Engrailed/Inv/GeneVI
complex in Drosophila SL-2 cells, where the Pc protein is exclusively
associated with transcriptionally silent genes, while Ph and Psc are present
irrespective of the transcriptional status
(Strutt and Paro, 1997).
Therefore the complete, `perfect' form of the class 2 PcG core complex may
mediate transcriptional repression more efficiently than form(s) lacking the
Rnf2 component. If this is the case, incorporation of the Rnf2 component into
the complex might be a limiting process to mediate transcriptional repression
and regulate its stability (Francis et
al., 2001). It is also possible that the role of Rnf2 is mediated
through its E3 ubiquitin ligase activity directed to histone H2A
(Wang et al., 2004a;
de Napoles et al., 2004).
Transcriptional repression of Hox genes in the developing embryo has been
shown to correlate with the association of Rnf2-containing class 2 PcG
complexes and H3-K27 trimethylation. De-repression of Hox genes in
Rnf2 and Suz12 mutant cells reveal the requirement of both
Rnf2 association and H3-K27 trimethylation in the mediation of this
transcriptional repression. As Rnf2 association to Hox genes is reduced in
Suz12 mutant ES cells and Rnf2 mutation alters Hox
expression without changing local levels of H3-K27 trimethylation, H3-K27
trimethylation mediated by class 1 PcG complexes at Hox genes may facilitate
subsequent binding of Rnf2-containing PcG complexes. Recruitment of
Rnf2-containing PcG complexes may in turn prevent the access of nucleosome
remodeling factors, such as SWI/SNF complex, leading to the formation of a
repressed chromatin status (Shao et al.,
1999; Levine et al.,
2002). Therefore, molecular circuitry underlying PcG silencing of
Hox genes seems to have been evolutionarily conserved between
Drosophila and mammals. It is also notable that Cbx2, a homologue of
Drosophila Pc, binds to Hoxb8 in transcriptionally active
embryonic tissues, despite the lack of histone H3 trimethylated at K27. This
is consistent with previous biochemical data that have shown the association
of purified or reconstituted PcG complexes with the nucleosomal templates
lacking histone tails (Shao et al.,
1999). The implication of these findings is that there are at
least two different means by which class 2 PcG complexes bind to the
chromatin, and that the association, which involves trimethylated H3-K27,
mediates the repression at the Hox genes in vivo
(Cao et al., 2002;
Czermin et al., 2002;
Muller et al., 2002).
The maintenance of regionally restricted expression of Hox genes is likely
to involve H3-K9 acetylation and H3-K4 methylation
(Milne et al., 2002;
Rastegar et al., 2004). We
have shown that these modifications of the histone tail increases
craniocaudally along the axis. Although the transcriptionally active posterior
tissues of 9.5 dpc and older embryos are more heavily acetylated at H3-K9 than
the anterior, non-Hox expressing tissues, some acetylation of H3-K9 at
Hoxb8 is seen in anterior regions where Hoxb8 expression is
repressed at early and later developmental stages. De-repression of
Hoxb8 expression upon depletion of Rnf2 in MEFs derived from the
cranial part of 9.5 dpc embryos suggests the involvement of Rnf2-containing
class 2 PcG complexes to mediate this transcriptional repression. Therefore,
our data suggest that the associations of Rnf2-containing PcG complexes and
acetylated H3-K9 may counteract each other and cooperate to maintain the
anterior boundaries of Hoxb8 expression at mid-gestational stages and
later. This is consistent with the antagonistic properties of Mll and
Bmi1 mutations (Hanson et al.,
1999). Moreover, the establishment of the differential binding of
the Rnf2 and H3-K9 acetylation at Hoxb8 during embryogenesis
temporally coincides with de-repression of that Hox gene in
Bmi1/Rnf110 and Phc1/Phc2 double
homozygotes, and loss of its transcription in Mll homozygotes
(Akasaka et al., 2001;
Yu et al., 1998;
Isono et al., 2005b).
Intriguingly, class 2 PcG complexes, which lack the Rnf2 component, are also
involved in the maintenance of H3-K9 acetylation in embryonic tissues where
Hox genes are expressed. This is consistent with predominant subnuclear
localization of several PcG proteins in the perichromatin compartment where
most pre-mRNA synthesis takes place (Cmarco et al., 2003). The molecular
mechanisms underlying this positive action remain unaddressed.
In conclusion, class 2 PcG gene products play distinct roles in embryonic territories, which are silent or active for Hoxb8 transcription, by forming complexes of different composition. Interaction between class 1 and class 2 PcG complexes mediated by trimethylated H3-K27 play decisive roles in Hox gene repression outside their expression domains, as seen in Drosophila. In addition, within the Hox expression domain, class 2 PcG complexes are involved in maintaining a transcriptionally active status, independent of H3-K27 trimethylation.
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Footnotes |
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Supplementary material for this article is available at http://dev.biologists.org/cgi/content/full/133/12/2371/DC1
* These authors contributed equally to this work 
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13-somite stage were dissected into
three pieces at the boundary between somites 9 and 10, and at the newly
generated somite boundary. WCE prepared from cranial region up to somite 9 (A)
and posterior unsegmented region (P) were subjected to ChIP analyses.
(D) Embryos at 9.5 dpc (about 25 somites) were bisected into anterior
(A) and posterior (P) pieces at the level of somite 9/10 boundary and
respective WCE were subjected to ChIP analyses. (E) Embryos at 10.5 dpc
(
