1 Centre of Electron Microscopy, University of Lausanne, 27 Bugnon, CH-1005
Lausanne, Switzerland
2 Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of
Amsterdam, P.O. Box 94062, 1090 GB Amsterdam, The Netherlands
* Author for correspondence (e-mail: sfakan{at}cme.unil.ch)
Accepted 15 October 2002
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Summary |
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Key words: PcG proteins, Gene silencing, Chromatin, Immunoelectron microscopy, Transcription
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Introduction |
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PcG proteins form multimeric complexes
(Franke et al., 1992;
Satijn and Otte, 1999a
;
Sewalt et al., 1998
;
Francis et al., 2001
;
Simon and Tamkun, 2002
) that
bind to specific genomic sites, named Polycomb responsive elements (PREs)
(Chan et al., 1994
;
Mihaly et al., 1998
;
Tillib et al., 1999
),
resulting in transcriptional silencing of nearby loci in a clonally stable
manner. Also, if PcG proteins are artificially targeted to a locus, it becomes
transcriptionally inactive (Alkema et al.,
1997
; Bunker and Kingston,
1994
; van der Vlag and Otte,
1999
). It is thought that PcG-mediated silencing involves changes
in chromatin structure (Paro et al.,
1998
; Singh, 1994
;
Singh and Huskisson, 1998
;
Simon and Tamkun, 2002
),
putatively inducing a heterochromatin-like structure
(Eissenberg and Elgin,
2000
).
The fine structural architecture of the cell nucleus has been extensively
studied for many years. These studies have allowed one to define several
structural compartments or domains in the nucleus and to approach the roles of
these domains in nuclear functions. It is now well established that the
perichromatin region is a site of DNA replication as well as of transcription
(for reviews, see Fakan, 1994;
Spector, 1996
;
Jaunin and Fakan, 2002
) and
that the perichromatin fibrils are in situ forms of nascent transcripts
(Fakan, 1994
;
Cmarko et al., 1999
).
Structural components occurring in the interchromatin space such as the
interchromatin granules or coiled (Cajal) bodies have been shown to be
involved in assembly and storage of pre-mRNA transcription factors
(Fakan, 1994
;
Spector, 1996
;
Puvion and Puvion-Dutilleul,
1996
; Misteli et al.,
1997
).
There is growing evidence that a tight relationship exists between
large-scale chromatin folding and gene activity
(Belmont et al., 1999;
Tumbar et al., 1999
;
Francastel et al., 1999
;
Lundgren et al., 2000
).
Transcriptionally active loci are located near the surface of compact
chromatin domains, possibly looping out towards the interchromatin compartment
(Fakan, 1994
;
Cmarko et al., 1999
;
Verschure et al., 1999
).
Buchenau et al. have presented evidence that PcG proteins in
Drosophila cells do not occur in compact heterochromatin
(Buchenau et al., 1998
).
Similarly, it has been shown that Drosophila PcG proteins
collectively bind to numerous sites on polytene chromosomes. These sites are
distinct from constitutive heterochromatin marked by heterochromatin protein 1
(HP1), suggesting that PcG proteins are not associated with heterochromatin
(Platero et al., 1995
;
DeCamillis et al., 1992
;
Rastelli et al., 1993
;
Sinclair et al., 1998
). In
contrast, Saurin et al. have shown that some cell types contain
nuclear-body-like domains that are enriched in PcG proteins and are associated
with pericentromeric heterochromatin
(Saurin et al., 1998
). Most
probably, PcG proteins do not silence loci by incorporating them into
constitutive heterochromatin, despite the idea that PcG-protein-mediated
silencing induces a heterochromatin-like structure
(Eissenberg and Elgin,
2000
).
Here we have investigated the subnuclear distribution of four human PcG proteins in two different human cell lines and in rat liver tissue using indirect immunolabelling in combination with light and electron microscopy. We find that each of the PcG proteins is concentrated in the perichromatin compartment, the same nuclear domain where the transcriptionally active genes are situated. This indicates that active and silenced genes are closely associated in space, suggesting that PcG-silencing is a local event, rather than affecting large chromatin domains. In addition to being associated with chromatin, PcG proteins also occur in the interchromatin region. It is not known whether this fraction is associated with looped-out chromatin or whether it is freely diffusing in the interchromatin space.
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Materials and Methods |
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Antibodies
The cells were immunolabelled with the following monoclonal (mouse) and
monospecific polyclonal (rabbit) primary antibodies against human PcG
proteins: mouse and rabbit anti-HPC2 (homologue of Drosophila
Polycomb) (Satijn et al.,
1997), rabbit anti-HPH1 (homologue of Drosophila
Polyhomeotic) (Gunster et al.,
1997
), rabbit anti-BMI1 (homologue of Drosophila
Posterior sex combs (Satijn et al.,
1997
; Satijn and Otte,
1999b
), rabbit anti-RING1 (a PcG protein associated polypeptide)
(Satijn et al., 1997
). For
double-labelling experiments, mouse anti-BrdU monoclonal antibody (Partec,
Münster, Germany) recognising also BrU in RNA was used. To visualise the
primary antibodies, goat anti-mouse and goat anti-rabbit secondary antibodies,
conjugated to fluorochromes (Jackson ImmunoResearch Laboratories, West Grove,
PA) or with different sizes of colloidal gold (Aurion, Wageningen, The
Netherlands, or Jackson ImmunoResearch Laboratories), were used.
Immunofluorescence microscopy
Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline
(PBS) for 10 minutes on glass coverslips. The fixed cells were permeabilised
with 0.5% Triton-X 100 (Sigma, St. Louis, MO/USA) in PBS and incubated with
PBS containing 100 mM glycine (Sigma) for 10 minutes. Subsequently, cells were
incubated overnight at 4°C with the primary antibodies. After washing in
PBS, cells were incubated with secondary antibodies for 1 hour at room
temperature and then washed with PBS. Cells were counterstained with DAPI at
room temperature. Slides were mounted in Vectashield (Vector, Burlingame, CA)
and were kept at 4°C until evaluation within 24 hours.
Images were recorded with a Zeiss LM510 confocal laser scanning microscope equipped with a 100x/1.23 NA oil immersion lens. A dual-wavelength argon ion laser was used to excite the two fluorochromes simultaneously. Pairs of images, showing the distribution of the antigen and of DAPI, were collected simultaneously. Optical sections through the middle of the nucleus were obtained at a scanning resolution of 512x512 pixels. Each image is the result of the averaging of 16 scans of the same optical section.
Immunoelectron microscopy
Chemical fixation or cryofixation protocols were used. Cells grown on glass
coverslips at 50-70% confluency were fixed with 4% paraformaldehyde in 0.1 M
Sörensen phosphate buffer, for 1 hour, at 4°C. T24 cells were
microinjected with BrUTP and cultured for 10 minutes at 37°C as described
previously (Cmarko et al.,
1999; Wansink et al.,
1993
), before fixation and further processing.
Pieces of rat liver were fixed in 4% paraformaldehyde immediately after
dissection, for 2 hours on ice. After washing with buffer, free aldehyde
groups were blocked by 0.5 M NH4Cl in PBS for 15 minutes on ice.
Cells and tissue were then dehydrated in ethanol, embedded in LRWhite resin,
which was allowed to polymerise for 24 hours at 60°C. To overcome some of
the potentially deleterious effects of the aldehydic fixation, some specimens
were treated using cryopreparative techniques. In short, cells grown on 14 mm
CellagenTM discs (ICN, Aurora, Ohio) were cryofixed by slam freezing
using a KF80 apparatus (Reichert, Vienna, Austria). The samples of rat liver
were fixed by high pressure freezing (Balzers Union, Balzers, Liechtenstein).
The frozen material was cryosubstituted in acetone using a Reichert CS Auto
cryosubstitution device and embedded into LRWhite resin
(von Schack and Fakan,
1993).
Ultrathin sections were mounted on Formvar/carbon-coated nickel grids and
processed for postembedding immunogold labelling, as described previously
(Cmarko et al., 1999). Briefly,
normal goat-serum-pretreated sections were incubated for 17 hours at 4°C,
with the primary antibody or, in the case of double labelling, with a mixture
of primary antibodies diluted in PBS with 0.05% Tween 20 (Sigma) and 0.1% BSA
(Fluka, Buchs, Switzerland). Colloidal gold-conjugated secondary antibodies
were diluted in PBS and reacted with sections for 30 minutes at room
temperature. The grids containing sections of chemically fixed cells were
submitted to regressive EDTA staining, which is preferential for nuclear
ribonucleoprotein constituents (Bernhard,
1969
), giving rise to a lower contrast of chromatin.
Alternatively, some grids were treated with a 0.2% osmium ammine solution for
1 hour at 20°C to specifically visualise DNA by a Feulgen-type reaction
(Cogliati and Gautier, 1973
).
In cryofixed and cryosubstituted material embedded in LRWhite resin and
conventionally contrasted with uranyl-lead staining, the chromatin tends to
have a bleached aspect and RNP constituents are well distinguished without the
EDTA treatment (von Schack and Fakan,
1993
).
In control specimens, sections were treated with the reaction mixture where the primary antibodies were omitted.
The grids were examined with a Philips CM 10 electron microscope at 80 kV using a 30-40 µm objective aperture.
Quantitative evaluation
For quantitative evaluation of the HPC2, BMI1 and RING1 ultrastructural
distribution in the nucleus of chemically fixed SW480 cells, 12 micrographs of
each group were analysed. Three nuclear regions were considered for
quantification: (i) condensed chromatin domains, (ii) the periphery of
condensed chromatin, that is, the perichromatin region representing the
interface between the condensed chromatin and the interchromatin region, and
(iii) the nucleoplasmic area free of condensed chromatin. The width of the
perichromatin region is not known and we set it at 40 nm (20 nm on each side
of the condensed chromatin edge) taking into account the resolution limits of
the indirect immunocytochemical labelling. The micrographs printed at a final
magnification of x52,500 were scanned and the surface area of each of
the above-mentioned nuclear regions was morphometrically determined, using the
Openlab measurement module (Improvision, Coventry, UK). The number of
individual gold particles (12 nm diameter) was counted by hand for each area
and the labelling density was expressed as the number of gold particles per
µm2.
Statistical analysis of the labelling density differences between the three
above-mentioned compartments in the same experimental group was performed
using the Wilcoxon signed ranks test
(Siegel and Castellan, 1988).
Statistical significance was set at P<0.01.
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Results |
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Visual inspection of fluorescence images further shows that, as expected at this spatial resolution, the nucleus seems to be completely filled with DAPI-stained material, that is, DNA. Only the nucleoli contain less DNA. Like DNA, the PcG proteins seem to be present throughout the nucleoplasm with the exception of the nucleoli. However, as the line scans in Fig. 1 show (panels E, J and M), there is no relationship between the detailed spatial distribution of DNA/chromatin (DAPI stained in Fig. 1C,G,I,L) and that of PcG proteins (Fig. 1A,B,F,H,K). Evidently, these four PcG proteins do not simply colocalize with chromatin domains. The resolution limit of light microscopy precludes a more detailed analysis of the localisation of PcG proteins with regards to the fine structure of chromatin areas and particularly of the perichromatin compartment. In order to investigate in more detail the subnuclear distribution of PcG proteins, we have carried out an ultrastructural study using immunoelectron microscopy.
Immunoelectron microscopic localisation of PcG proteins
Sections of cultured cells and rat liver tissue were prepared for electron
microscopy either after aldehyde fixation or following cryofixation and
cryosubstitution in the absence of chemical fixatives. Particularly the latter
method, applied to rat liver tissue, exhibited very good preservation of
nuclear fine structure. Immunolabelling of nuclear constituents on ultrathin
sections of intact cells is the method of choice for high-resolution in situ
localisation of cellular components. In contrast to methods that involve
treatment of the specimen with detergents, this procedure avoids protein
extraction and possible displacement of antigens in the cell. Moreover,
immunolabelling on sections of embedded material circumvents the problem of
limited accessibility of antibodies into compact cellular compartments, such
as condensed chromatin, because the immunoreaction takes place on the surface
of physically sectioned material.
Cell and tissue sections were labelled with antibodies against four human PcG proteins: HPC2, HPH1, BMI1 and RING1. Each of five anti-PcG protein antibodies that were used for immunoelectron microscopy gave a significant signal in the cell nucleus. After EDTA regressive staining of sections, condensed chromatin is relatively electron translucent compared with the RNP-containing perichromatin fibrils and interchromatin compartment (Fig. 2). By contrast, DNA-specific osmium ammine staining resulted in well-contrasted electron dense chromatin areas, leaving the interchromatin domain unstained (Fig. 3A, Fig. 4A).
|
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The immunogold labelling of PcG proteins in paraformaldehyde-fixed material showed intense labelling of the border of condensed chromatin areas, that is, the perichromatin region. The interchromatin compartment was also significantly labelled. Strikingly, almost no label was observed inside condensed chromatin domains (Table 1; Fig. 2, Fig. 3A). Double labelling experiments with the monoclonal anti-HPC2 antibody and rabbit antibodies against the other PcG proteins showed similar distributions for the different PcG proteins, although we did not observe frequent close colocalisation of the two markers (Fig. 4A). This can be explained by steric hindrance occurring within relatively small closely packed molecular assemblies.
|
The above-described distribution of PcG proteins observed in chemically
fixed cells was confirmed on cryofixed samples. In cultured cells and in rat
hepatocytes the proteins were present predominantly at the periphery of
condensed chromatin or throughout the interchromatin space, frequently
associated with RNP fibrils (Fig.
3B). The perichromatin area, previously shown to be the major site
of nucleoplasmic RNA synthesis (Cmarko et
al., 1999; Fakan et al.,
1976
), contains a large number of perichromatin fibrils, which are
the in situ forms of nascent heterogeneous RNA transcripts
(Fakan, 1994
). Each of the
four PcG proteins is occasionally observed in close contact with a
perichromatin fibril (arrowheads in Fig.
2). This is confirmed by dual labelling of PcG proteins and of
nascent RNA, which was tagged by a brief in vivo incorporation of
microinjected BrUTP. Nascent RNA and PcG proteins both occurred in the
perichromatin regions and occasional close association of the two signals
within this nuclear domain was observed
(Fig. 4B). The intranuclear
distribution of PcG proteins in the human cell lines and in rat liver cells
was similar. Each of the four PcG proteins was also abundantly present in the
interchromatin compartment, which is virtually devoid of chromatin. There, PcG
proteins were often localised on the periphery of clusters of interchromatin
granules whereas the internal area of these clusters remained devoid of label
(Fig. 4B). Most probably, the
PcG proteins in the perichromatin region are part of multi-protein silencing
complexes, whereas those in the interchromatin space may represent a freely
diffusing pool of protein molecules.
In control assays, where the primary antibody was omitted, the level of labelling over cell sections was negligible. Similarly, the background labelling observed over the resin outside the cells was very low.
Quantitative evaluation of immunogold labelling distribution
Quantitative evaluation of the labelling within condensed chromatin, the
perichromatin region (here defined as a 40 nm rim around condensed chromatin
domains) and the interchromatin space demonstrates that PcG proteins are
highly concentrated in the perichromatin region
(Table 1). Only a few gold
particles were found in condensed chromatin domains. The differences in
labelling densities between the three nuclear compartments are statistically
significant (Wilcoxon signed rank test, P<0.01).
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Discussion |
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To further explore the relationship between gene activity and epigenetic
gene silencing on the one hand and chromatin organisation on the other hand,
we have analysed the localisation of four members of the human PcG protein
family in the cell nucleus. PcG proteins were originally identified through
their involvement in the epigenetic silencing of homeotic genes in
Drosophila (see Pirrotta,
1998). More recently, it has been shown that PcG proteins are
evolutionarily conserved and play an important role in the epigenetic
repression of many other genes in higher eukaryotes, including plants and
animals (Jacobs and van Lohuizen,
1999
; Satijn and Otte,
1999a
). PcG protein complexes bind to genomic sequence elements
called Polycomb response elements (PREs), thus repressing nearby genes in cis
(Eissenberg and Elgin, 2000
;
Lyko and Paro, 1999
;
Jacobs and van Lohuizen,
1999
). Here, we investigated in two different human cell lines and
in rat liver tissue the subnuclear distribution of four human PcG proteins,
that is, HPC2, HPH1, BMI1 and RING1, with respect to the distribution of
condensed chromatin and sites of transcription. We carried out indirect
immunofluorescent and immunogold electron microscopic labelling of these
proteins. Condensed chromatin was visualised by counterstaining with DAPI in
confocal light microscopy and using the osmium ammine staining for DNA or the
EDTA regressive staining in electron microscopy.
Our results for all four PcG proteins are identical. The four antigens were
almost exclusively localized in the nucleus. Unexpectedly, HPC2, HPH1, BMI1,
RING1 proteins were found highly concentrated in the perichromatin
compartment, representing the border of condensed chromatin domains
(Table 1). In addition, the PcG
proteins also occur in the interchromatin space although at a five- to
ten-fold lower concentration (Table
1), possibly as freely diffusing protein molecules. Strikingly,
PcG proteins were almost absent from condensed chromatin
(Table 1). This lack of label
is not due to inaccessibility of antigens in compact chromatin, because
labeling takes place on the surface of physically sectioned specimens. Our
results show that PcG proteins are preferentially located in the perichromatin
compartment. This compartment also contains transcriptionally active genes
(Cmarko et al., 1999;
Verschure et al., 1999
), as
further confirmed by dual labeling of PcG proteins with nascent RNA
(Fig. 4B). These results
strongly suggest that loci silenced by PcG proteins are spatially interspersed
with transcriptionally active genes, both in space, that is, in the
perichromatin compartment, and probably also on the linear genome. We conclude
that PcG proteins act only locally rather than involving larger chromatin
domains.
Our findings argue against a mechanism in which silencing of genes by
binding PcG proteins to PREs gives rise to repositioning of silenced loci
inside compact chromatin domains, that is, away from the perichromatin
compartment. Clearly, the state of chromatin after silencing by PcG proteins
is different from that in heterochromatin that seems to be involved in
silencing loci by, for instance, Ikaros-related gene product
(Brown et al., 1997;
Fisher and Merkenschlager,
2002
) or by inactivation of certain enhancer functions
(Francastel et al., 1999
).
The present findings, revealing that PcG proteins are preferentially
associated with the periphery of condensed chromatin and therefore probably
exert their gene silencing function in the same compartment that contains
transcriptionally active chromatin, are in agreement with observations showing
that PcG proteins occupy different positions on polytene Drosophila
chromosomes than HP1, a typical marker for constitutive heterochromatin
(Platero et al., 1995;
Sinclair et al., 1998
).
Our data also agree with the previous suggestion, based on light
microscopic observations, that the distribution of PcG proteins does not
correlate with local high concentrations of DNA
(Buchenau et al., 1998) and
that PcG proteins occur in a punctated pattern
(Buchenau et al., 1998
;
Dietzel et al., 1999
).
However, in contrast to the conclusion of Buchenau et al.
(Buchenau et al., 1998
), we
demonstrate that PcG proteins accumulate in the same compartment where
transcription takes place. This discrepancy is probably due to the fact that
the above authors used the hnRNP-K protein as a marker for transcription sites
rather then nascent RNA.
We observed that each of the four PcG proteins is occasionally associated
with RNP fibrils in perichromatin regions and interchromatin space (arrowheads
in Fig, 2,
Fig. 3B). It suggests that PcG
proteins may interact with RNA-containing nuclear components. This is in line
with a recent finding suggesting that chromodomain proteins preferentially
interact with RNA (Akhtar et al.,
2000).
These and earlier observations raise important questions about higher order chromatin folding in relation to gene transcription and gene silencing. The emerging picture is that in interphase nuclei chromatin is organised in such a way that active genes, as well as PcG silenced loci, are localised at the periphery of the compact chromatin domain. This seems to impose severe constraints on the way the chromatin fibre is folded in vivo. It further raises the question as to what sequences are inside compact chromatin domains. Is there only repetitive, non-coding DNA or are there also coding sequences that are silenced by mechanisms that do not involve PcG proteins? Present work is focusing on these questions.
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Acknowledgments |
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