1 John Innes Centre, Colney, Norwich NR4 7UH, UK
2 Instituto Superior de Agronomia, Tapada da Ajuda, Lisbon, Portugal
3 Institute for Molecular Biotechnology RWTH, Warringer Weg 1, D-52074 Aachen,
Germany
* Author for correspondence (e-mail: peter.shaw{at}bbsrc.ac.uk)
Accepted 5 September 2002
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Summary |
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Key words: Interphase chromosomes, Transgenes, DNA methylation, Histone acetylation, Chromatin remodelling
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Introduction |
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One current model for higher-order chromosome organisation suggests that
active genes are located on loops that may be mobile within the nucleus
(Cook, 1995). It has
furthermore been suggested that these loops may be pulled through fixed
polymerases during transcription and replication, rather than the polymerases
moving along fixed DNA strands (Dickinson
et al., 1990
; Jackson et al.,
1993
; Iborra et al.,
1996
; Cook, 1999
).
The position of certain individual genes has been associated with
transcriptional state. For example, the ikaros proteins are located around
centromeric heterochromatin in lymphocytes, and silenced genes are recruited
to the ikaros sites, whereas active genes are located away from
heterochromatin (Brown et al.,
1999
). In yeast, the Sir-dependent telomeric gene silencing is
associated with recruitment of the silenced genes to Sir protein sites at the
nuclear periphery (Gasser et al.,
1998
; Laroche et al.,
2000
). Francastel et al. showed that transcriptional enhancers may
suppress silencing of a transgene by preventing its localisation close to
centromeric heterochromatin and recruiting it into an active compartment
(Francastel et al., 1999
);
mutations in the enhancer that led to increased silencing resulted in
localisation at the centromeric heterochromatin. Dernburg et al. showed that
insertion of heterochromatin at one allele of the brown locus in
Drosophila caused both the altered gene and the other allele to
associate with centromeric heterochromatin
(Dernburg et al., 1996
).
Lundgren et al. used a transgene integrated into pericentromeric
heterochromatin to show that transcriptional activation was associated with
changes in the heterochromatin structure, but the transgene was not relocated
away from the heterochromatin on activation
(Lundgren et al., 2000
).
Tumbar and Belmont used GFP in vivo labelling to show that the VP16
transcriptional activator can alter the nuclear positioning of an engineered
reporter sequence (Tumbar and Belmont,
2001
).
Changes in chromatin organisation are associated with transcriptional
activation, DNA methylation and histone acetylation. Several chromatin
remodelling complexes have been identified, in plants as in animals, of which
the best known are the SNF2 type
(Felsenfeld, 1996;
Workman and Kingston, 1998
;
Travers, 1999
), and these are
also specifically involved in transcriptional regulation. In plants, DNA
methylation has been correlated with chromatin structure by the identification
of the ddm1 gene from Arabidopsis thaliana, which encodes an
SNF2-family protein (Jeddeloh et al.,
1999
). Mutants defective in this gene gradually lose their normal
methylation patterns, with repetitive regions of the genome being
hypomethylated first. This suggests that chromatin conformational changes are
necessary to allow access of maintenance methylases during DNA
replication.
DNA methylation and histone acetylation have been clearly linked with the
regulation of gene expression, with active genes tending to be
under-methylated and associated with nucleosomes whose core histones show
increased acetylation (Loidl,
1994; Workman and Kingston,
1998
; Wolffe and Matzke,
1999
; Wolffe and Guschin,
2000
). Current hypotheses suggest that histone acetylation
produces a more open chromatin conformation, which allows greater access for
the transcriptional machinery. There is evidence going back over many years
that increased histone acetylation is correlated with increased transcription
(Allfrey et al., 1964
;
Kouzarides, 2000
), and in the
past few years several specific histone acetylases and deacetylases have been
identified in animals, plants and fungi
(Cheung et al., 2000
;
The Arabidopsis Genome Initiative,
2000
). DNA methylation and histone deacetylation have been
directly functionally linked. For example, the transcriptional repressor
proteins MeCP1 and MeCP2 have methyl-CpG-binding domains and independently or
together interact with other structural components of the chromatin to
regulate transcription (Meehan et al.,
1992
; Meehan et al.,
1989
; Ng and Bird,
1999
). MeCP2 appears to act by recruiting histone deacetylases
(HDAcs) (Nan et al., 1998
;
Jones et al., 1998
), and it
can displace H1 from the nucleosome (Nan
et al., 1996
).
There is as yet little direct evidence to link the molecular-scale changes in DNA accessibility or conformation accompanying transcriptional activation, DNA methylation and histone acetylation to the higher-order organisation seen at the level of interphase chromosomes. We have made use of the very regular organisation seen in wheat interphase chromosomes to analyse how they are modified by global changes in methylation or histone acetylation. We show that after germinating seeds either in the presence of 5-Azacytidine (5-AC), leading to DNA hypomethylation, or trichostatin A (TSA), which results in histone hyperacetylation, the architecture of the interphase chromosome territories shows significant changes. We have also used transgenic wheat lines to investigate changes in chromosome architecture at the level of individual genes. In lines carrying multiple transgene integrations at widely spaced sites, we show that the transgenes are usually colocalised during interphase, but that after 5-AC or TSA treatment the transgene activity increases, and the multiple transgene sites are dispersed. This suggests that the colocalisation/dispersion of the transgenes may be a function of specific interphase chromosome architecture.
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Materials and Methods |
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Protein extraction and immunoblotting analysis
Total root proteins were extracted by homogenising roots in SDS sample
buffer (Laemmli, 1970) [Sample
buffer: 0.125 M TRIS/HCl pH 6.8, 4% (w/v) SDS, 20% glycerol, 10% (v/v)
2-mercaptoethanol, 0.002% (w/v) bromophenol blue]. Protein samples were
resolved by SDS gel electrophoresis on 15% gels and transferred to
nitrocellulose by western blotting (Towbin
et al, 1979
). The blots were probed with antibody AHP418
(Serotec), which is specific for acetylated histone H4, or antibody AHP416
(Serotec), which is specific for Histone H4 acetylated at lysine 12, diluted
in TBS according to the manufacturer's instructions. Proteins were visualised
using a secondary antibody goat anti-rabbit alkaline phosphatase, diluted 1 in
1000 in TBS.
Root sections
30 µm thick sections from root tips were sectioned using a Vibratome
Series 1000 (TAAB Laboratories Equipment Ltd., Aldermarston, UK) and allowed
to dry on multi-well slides (ICN Biomedicals Inc.). The slides were
pre-treated by washing in 3% (v/v) Decon for 1 hour, rinsing thoroughly with
distilled water. They were then coated with a freshly prepared solution of 2%
(v/v) 3-aminopropyl triethoxy silane (APTES, Sigma) in acetone for 10 seconds
and activated with 2.5% (v/v) glutaraldehyde in phosphate buffer for 30
minutes, rinsed in distilled water and air dried.
In situ hybridisation on wheat root sections
The tissue sections were permeabilised by incubation with 2% (w/v)
cellulase (Onozuka R-10) in TBS for 1 hour at room temperature, washed in TBS
for 10 minutes, dehydrated in an ethanol series of 70% and 100% and air dried.
Root sections from wheat transgenic lines were additionally treated with RNAse
(100 µg/ml) for 1 hour at 37°C, washed in 2xSSC (20xSSC: 3
M sodium chloride, 300 mM trisodium citrate, pH 7.0) and dehydrated as
described above. Genomic in situ hybridisation and generation of total genomic
probe was performed according to Schwarzacher et al.
(Schwarzacher et al., 1992)
and Abranches et al. (Abranches et al.,
1998
). The hybridisation mixture contained 50% deionised
formamide, 20% dextran sulphate, 0.1% sodium dodecyl sulphate, 10%
20xSSC, 200 ng of rye genomic DNA sonicated to 10-12 kb fragments as a
probe and 1 µg of sonicated salmon sperm as blocking DNA. Fluorescence in
situ hybridisation was used to visualise the transgenes on wheat root
sections, using pHAC25 DNA (200 ng) as a probe. Probes were labelled with
digoxigenin-11-dUTP (Boehringer Mannheim Corp. Indianapolis, IN) or
biotin-16-dUTP (Boehringer Mannheim) by nick translation. Denaturation of the
hybridisation mixture was carried out at 95°C for 5 minutes, cooled in ice
for another 5 minutes and immediately applied to the sections. Target DNA
denaturation was carried out in a modified thermocycler (Omnislide; Hybaid
LTD., Long Island, NY) at 78°C for subsequent hybridisation at 37°C
overnight. Post-hybridisation washes were carried out using 20% formamide in
0.1SSC at 42°C.
BrUTP incorporation into tissue sections
For transcription analysis, the procedures followed are those described
previously (Thompson et al.,
1997; Abranches et al.,
1998
). Briefly, vibratome sections were cut in a Modified
Physiological Buffer (MPB: 100 mM potassium acetate, 20 mM KCl, 20 mM Hepes; 1
mM MgCl2; 1 mM ATP (disodium salt, Sigma) in 50 mM Tris, pH 8.1%
(v/v); 1% (v/v) thiodiglycol (Sigma), 2 µg/ml aprotinin (Sigma) and 0.5 mM
PMSF (Sigma). To improve nuclear transcription as opposed to nucleolar
transcription, 1% BSA was added to the MPB buffer. The tissue sections were
transferred to a tissue-handling device
(Wells, 1985
) for subsequent
ease of handling. The permeabilisation was done by a very brief treatment (10
seconds) with 0.05% Tween 20 in MPB. The transcription mix consisted of 50
µM CTP (sodium salt, Pharmacia), 50 µM GTP (sodium salt, Pharmacia), 25
µM BrUTP (sodium salt, Sigma), 125 µM MgCl2, pH 7.4 with KOH); 100 U/ml
RNA guard (Pharmacia) made up in MPB. The tissue was incubated with the
transcription mix for 5 minutes and then fixed in 4% formaldehyde in PEM as
described above. After fixation, the sections were washed in TBS, then in
water and finally removed from the tissue-handling device and placed onto
activated APTES-treated slides.
Immunodetection
Probes labelled with digoxigenin were detected by an anti-digoxigenin
antibody conjugated to FITC (Boehringer Mannheim Corp., Indianapolis, IN), and
biotin-labelled probes were detected with extravidin-cy3 (Sigma, Chemical
Co.). Both antibodies were diluted in 3% BSA in 4xSSC/ 0.2% tween-20
(Sigma), and the antibody incubations were carried out in a humid chamber for
1 hour at 37°C followed by 3x5 minutes washes in 4xSSC/0.2%
Tween-20 at room temperature. The detection of BrUTP incorporation involved
incubation for 1 hour at room temperature with mouse anti-BrdU (Boehringer)
followed by a second incubation with a secondary fluorescent anti-mouse
Alexa-568 (Molecular Probes) antibody for 1 hour at room temperature. The
sections were counterstained with 1 µg/ml,
4'6-diamidino-2-phenylindole (DAPI Sigma Chemical Co) for 5
minutes and mounted in Vectashield antifade solution (Vector Laboratories Inc.
Burlingame, CA).
ß-Glucuronidase (Gus) assay
Gus activity was determined by testing root material by a quantitative
assay as described previously (Jefferson
et al., 1987), using 4-methyl umbelliferyl glucuronide (MUG) as a
substrate.
Confocal fluorescence microscopy and imaging processing
Confocal optical section stacks were collected using a Leica TCS SP
confocal microscope (Leica Microsystems, Heidelberg GMbH, Germany) equipped
with a Krypton and an Argon laser. The microscopy data were then transferred
to NIH image (a public domain program for the Macintosh by W. Rasband
available via ftp from
ftp://zippy.nimh.nih.gov)
and composited using Adobe Photoshop 5.0 (Adobe Systems Inc., Mountain View,
CA). 3D models were made from stacks of confocal sections using Object-Image
[an extension to NIH image written by Vischer et al.
(Vischer et al., 1994)] by
drawing manually the limit of the nucleus and marking the localisation of the
transgene fluorescence sites as dots. The 3D reconstruction models were
visualised using Rotater (by Craig Kloeden) available from
ftp://Rarn.adelaide.edu.au/rotater/rotater-3.5.cpt.hqx).
Final images were printed on a Pictography P3000 printer.
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Results |
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Induced changes in DNA methylation or histone acetylation cause
remodelling of interphase chromosome territory organisation
To determine whether changes in the DNA methylation or histone acetylation
state caused observable large-scale changes in interphase chromosome
organisation, we germinated the seedlings in the presence of either
5-azacytidine (5-AC), which reduces DNA methylation
(Neves et al., 1995;
Castilho et al., 1999
), or
Trichostatin A (TSA), which inhibits histone deacetylases, thus increasing
histone acetylation levels (Yoshida et
al., 1990
). In pilot experiments we germinated seedlings on a
range of concentrations of the two drugs, and chose for further analysis the
highest concentration in each case, which allowed near normal growth and
development (80 µM 5-AC, or 15 µM TSA). In both cases, higher
concentrations substantially reduced growth, and still higher concentrations
were lethal. The concentrations used for further study gave growth rates of at
least 90% of that shown by the control seedlings germinated in water. Castilho
et al. have shown directly by analysis with methylation-sensitive restriction
enzymes that germination of wheat seedlings in the presence of 5-AC does
indeed decrease the level of DNA methylation
(Castilho et al., 1999
). For
the TSA treatment, western blotting using antibodies specific for acetylated
histone H4 showed that germination of seedlings in the presence of TSA caused
biochemically detectable increases in histone acetylation
(Fig. 3). Since in animal cells
TSA has been reported to cause cell cycle arrest at G1 and G2
(Yoshida and Beppu, 1988
), we
used flow cytometric analysis to measure the relative DNA content of wheat
nuclei extracted from roots germinated in water or TSA. No significant changes
were seen in the relative numbers of G1, S and G2 nuclei, showing that there
was little effect on the cell cycle using this TSA concentration (data not
shown). After treatment with either of these reagents, there were striking
changes in the interphase chromosome organisation. The two chromosome arms,
which in the controls usually lay so close together as to be indistinguishable
(Fig. 1A,
Table 1), became widely
separated (Fig. 1B-E,
Table 1). Sometimes, but not
always, the two telomeres remained close to each other, whereas the rest of
the chromosome arms lay apart. The overall Rabl configuration was, however,
maintained, with telomeres and centromeres remaining on opposite sides of the
nuclear envelope.
|
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The results from a quantitative analysis of many nuclei from different seedling roots are shown in Table 1. The frequency of colocalisation of the arms is very high in untreated seedlings - 89% and 74% in 1R and 5R chromosomes, respectively. We also identified some cases in which the two interphase chromosome arms were of different lengths such as the chromosome 5R, which in metaphase has a sub-metacentric morphology. The complete separation of the two arms was very rare in the control seedlings (4% and 2% in 1R and 5R chromosomes, respectively). On the other hand, in seedlings treated with 5-AC and TSA, the interphase chromosome arms in each chromosome were often found separated from each other (44% and 48% in 1R and 5R chromosomes, respectively, in seedlings germinated in 5-AC; 22% and 8% in 1R and 5R, respectively when the seedlings were germinated in TSA).
To analyse the effects on individual chromosome arms in more detail we used single arm translocation lines. With both 5-AC (Fig. 2D,E) and TSA (Fig. 2F,G) the labelling pattern of the chromosome arms became irregular and showed gaps where the DNA was decondensed. The effect of 5-AC was more dramatic, and the arms were dispersed into several smaller labelled regions separated by gaps, corresponding to chromosome regions where the chromatin was decondensed. The arms showed a more extended, meandering path across the nucleus. With TSA, the arms typically showed about five regions with intervening gaps of decondensed chromatin. However, the chromosome arms appeared to remain smoother in appearance, straighter and generally closer to their normal appearance than with 5-AC.
Induced changes in DNA methylation or histone acetylation alter the
relative positions of individual genes during interphase
In order to analyse the positions of specific genes we used transgenic
wheat lines made by particle bombardment with a plasmid containing the GUS
reporter gene. Fluorescence in situ hybridisation on intact root tissue slices
was used to visualise the transgene arrangement in interphase nuclei. Three
lines were analysed in detail [line numbers refer to the original
characterisation, given in (Abranches et
al., 2000)]. Line 6 is homozygous, and carries five transgene
copies on each homologue of chromosome 4A. In situ labelling on metaphase
chromosomes shows two integration sites on opposite arms of the chromosome,
one in a sub-telomeric position on the short arm and the other about one-third
of the arm length from the telomere of the long arm of the chromosome
(Abranches et al., 2000
). Line
2, which is not homozygous, contains more than 10 copies of the transgene at
four distinct sites along the short arm of chromosome 6B, spanning 30% of the
length of the short arm of the chromosome
(Abranches et al., 2000
). All
the line 2 plants analysed in this paper were heterozygous, since homozygous
plants have a strong tendency to silence. Finally, line 3 is homozygous and
carries two transgenes per homologue at a single site at metaphase on the long
arm of chromosome 6B (Abranches et al.,
2000
).
Despite being well separated along the metaphase chromosomes, in these
lines the multiple transgene sites are brought into close physical proximity
during interphase (Abranches et al.,
2000). To quantify the transgene arrangements in detail, confocal
section stacks were modelled using Object-Image
(Vischer et al., 1994
), by
tracing the outline of the nuclei on each section and marking every
distinguishable transgene site with a red dot, as shown in
Fig. 4.
|
Fig. 4A shows a single confocal section of a typical group of cells from a control seedling of line 6, and Fig. 4D shows the corresponding 3D models. Only two FISH signals per nucleus are seen in these nuclei, that is, a single site per homologue, showing that the sites on opposite chromosome arms are located closer together than the optical resolution limit. When seedlings were germinated in either 5-AC or TSA, we observed a greater number of transgene sites, more closely correlating with the number of sites seen on the metaphase chromosomes. Thus line 6 germinated in 5-AC (Fig. 4B,E) showed mostly four sites per nucleus (a,b,c): the G2 nucleus (d) shows eight sites. Fig. 4C,F shows the equivalent results with TSA again all the four nuclei show four sites. Fig 4G shows a group of nuclei from a control seedling of line 2, and in Fig. 4J the corresponding 3D models are shown. A single FISH signal is shown in nuclei a,c,d and two signals in nucleus b. Fig. 4H,K shows nuclei from line 2 germinated in 5AC. Four FISH signals are shown in nucleus a and three or two FISH signals are shown in nuclei b and c, respectively. Fig. 4I,L show nuclei from line 2 germinated in TSA. Four FISH signals are shown in nuclei a,d, three in nucleus c and two in nucleus b. To obtain reliable quantitative data, we modelled several hundred nuclei from the three lines using the three germination conditions. Only clearly labelled, intact nuclei were included in the analysis, and large nuclei clearly in the G2 phase of the cell cycle were excluded. The number of sites seen in the various lines is shown in Table 2. This confirms quantitatively the results shown in Fig 4. In order to be able to compare the results for the homozygous plants with those from the heterozygous line 2 plants, which carry the transgenes on only one homologue, we have presented the results in Table 2 as counts per (interphase) chromosome. In all the nuclei from the homozygous lines included in the analysis, the transgene sites were arranged in two well separated groups corresponding to the two homologous chromosomes. In line 6, the majority of interphase chromosomes (84%) showed a single site in the control seedlings, whereas only 16% showed two or more sites. Germinated on either 5-AC or TSA, only about 20% showed a single site, and the remainder showed two or more sites. The mean number of sites per homologue increased from 1.2 in the controls to 1.8 or 1.9 after 5-AC or TSA, respectively. Line 3, which show only a single transgene site at metaphase, showed a single site per homologue whether germinated on water alone or in the presence of 5-AC or TSA.
|
In heterozygous plants from line 2, 95% of interphase chromosomes showed one or two sites, with a mean of 1.5. This increased to means of 2.0 or 3.0 germinated on 5-AC or TSA respectively.
Induced changes in DNA methylation or histone acetylation cause an
increase in transgene activity
Histochemical staining and a fluorometric assay were used to determine GUS
activity in roots in control seedlings germinated in water and in seedlings
germinated in the presence of either 5-AC or TSA.
Table 3 shows the results from
the quantitative fluorometric assay on the three transformed lines and the
untransformed control. A striking result was that 5-AC treatment substantially
increased activity in all the transformed plants. TSA increased the activity
in line 6 and line 2 but not to the same extent as 5-AC. A conclusion of these
measurements is that these lines, which all contain multiple inserts, may all
be partially transcriptionally suppressed.
|
Induced changes in DNA methylation or histone acetylation do not
cause obvious changes in the distribution of transcription sites in the
nucleus
BrUTP incorporation into unfixed wheat root sections was used to determine
the organisation of transcription sites
(Fig. 5). As described
previously, this labelling shows many closely spaced punctate sites in the
nucleolus (Thompson et al.,
1997), and more dispersed foci in the nucleoplasm
(Abranches et al., 1998
). This
distribution was confirmed in control seedling roots germinated in water
(Fig. 5A). In spite of the
large-scale reorganisation of the chromosome territories caused by 5-AC or TSA
treatment, there was no obvious difference in the overall organisation of
transcription sites visualised by BrUTP after either treatment. A similar
punctate labelling was seen in the nucleoplasm
(Fig. 5B,C).
|
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Discussion |
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We have further characterised the changes in nuclear organisation by using
transgenic wheat lines carrying multiple, widely separated transgene
insertions. During interphase in these lines, the multiple transgenes show a
strong tendency to colocalise during interphase
(Abranches et al., 2000). Line
6 has a total of approximately five transgene copies at two sites on opposite
chromosome arms, and line 2 has about 10 copies located at four sites
extending along approximately one-third of the metaphase chromosome arm, and
thus separated by several megabases of genomic sequence. In each case, a
simple model in which the metaphase chromosome is folded in half at the
centromere and extended across the nucleus in interphase would place these
sites 4-6 µm apart (Abranches et al.,
2000
). Thus specific aspects of the interphase chromosome
structure or direct, ectopic interactions between the sequences or proteins
bound to them must cause these sites to colocalise.
The GUS activity of all the transgenic lines we analysed was increased by 5-AC, and in most cases was also increased by TSA, suggesting that these lines, which all show colocalisation of more than one transgene copy, are all at least partially suppressed. It will be interesting to determine whether this is due to complete silencing of a subset of the transgenes in each nucleus or a partial suppression of all of them.
After germination in either 5-AC or TSA, the transgenes were more dispersed in the interphase nuclei and were generally seen as clusters of separate sites rather than single sites. As a control, we used a line that shows a single site at metaphase, and this line showed a single site per homologue in interphase as predicted, after germination on water or in the presence of either 5-AC or TSA. If the multiple sites are colocalised in these transgenic plants because of specific chromosome architecture, then the dispersal of the sites can be explained by the disruption of chromosome territory structure that we have shown occurs after these treatments. If this is the case, it implies that aspects of the interphase chromosome architecture are very well defined, and are maintained or reproduced through many cycles of cell division. On the other hand, if the colocalisation is a consequence of ectopic pairing, or interaction with specific nuclear structures, then such interactions must themselves be modified by the changes in DNA methylation or histone acetylation. Further work analysing the behaviour of the genomic sequences flanking the transgenes will be needed to distinguish between these possibilities.
Although we have shown extensive rearrangement of interphase chromosome territories, and a dispersal of individual transgene sites after TSA or 5-AC treatment, we saw little change in the overall organisation of the nuclear transcription sites revealed by BrUTP incorporation. This suggests that there is some other organisational principle underlying the location of the transcription sites and is consistent with the presence of specialised nuclear locations to which active genes are recruited.
There is now a great deal of evidence that genes can be sequestered to
transcriptionally inactive regions of the nucleus as part of their regulation
(Cockell and Gasser, 1999).
Telomeric silencing has been widely studied in yeast (e.g.
Gasser et al., 1998
), and
there are now several examples of sequestering inactive genes to centromeric
heterochromatic regions in mammalian cells
(Brown et al., 1999
;
Dernburg et al., 1996
;
Francastel et al., 1999
;
Lundgren et al., 2000
).
Although our results indicate that specific genes can be repositioned by large
distances in molecular terms (
4-6 µm), the regular, polarised
organisation of the interphase chromosomes in wheat means that a large
proportion of all the chromosome arms in these nuclei must be further away
than this from either the centromeres or the telomeres, which are invariably
located at the nuclear periphery. It is possible that a level of
transcriptional control equivalent to that already observed at telomeric or
centromeric heterochromatin in mammalian and yeast nuclei may be achieved by
sequestering genes to the heterochromatic regions interspersed along these
large plant chromosomes.
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Acknowledgments |
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References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Abranches, R., Beven, A. F., Aragon-Alcaide, L. and Shaw, P.
(1998). Transcription sites are not correlated with chromosome
territories in wheat nuclei. J. Cell Biol.
143, 5-12.
Abranches, R., Santos, A. P., Wegel, E., Williams, S., Castilho, A., Christou, P., Shaw, P. and Stöger, E. (2000). Widely separated multiple transgene integration sites in wheat chromosomes are brought together at interphase. Plant J. 24,713 -723.[CrossRef][Medline]
Allfrey, V. G., Faulkner, R. and Mirsky, A. E. (1964). Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc. Natl. Acad. Sci. USA 61,786 -794.
Brown, K. E., Baxter, J., Graf, D., Merkenschlager, M. and Fischer, A. G. (1999). Dynamic repositioning of genes in the nucleus of lymphocytes preparing for cell division. Mol. Cell. 3,207 -217.[Medline]
Castilho, A., Neves, N., Rufini-Castaglione, M., Viegas, W. and
Heslop-Harrison, J. S. (1999). 5-methylcytosine distribution
and genome organization in Triticale before and after treatment with
5-azacytidine. J. Cell Sci.
112,4397
-4404.
Cheung, W. L., Briggs, S. D. and Allis, C. D. (2000). Acetylation and chromosomal functions. Curr. Opin. Cell Biol. 12,326 -333.[CrossRef][Medline]
Cockell, M. and Gasser, S. M. (1999). Nuclear compartments and gene regulation. Curr. Opin. Genet. Dev. 9,199 -205.[CrossRef][Medline]
Cook, P. R. (1995). A chromomeric model for
nuclear and chromosome structure. J. Cell Sci.
108,2927
-2935.
Cook, P. R. (1999). The organization of
replication and transcription. Science
284,1790
-1795.
Croft, J. A., Bridger, J. M., Boyle, S., Perry, P., Teague, P.
and Bickmore, W. A. (1999). Differences in the localization
and morphology of chromosomes in the human nucleus. J. Cell
Biol. 145,1119
-1131.
Dernburg, A. F., Broman, K. W., Fung, J. C., Marshall, W. F., Philips, J., Agard, D. A. and Sedat, J. W. (1996). Perturbation of nuclear architecture by long- distance chromosome interactions. Cell 85,745 -759.[Medline]
Dickinson, P, Cook, P. R. and Jackson, D. A. (1990). Active RNA polymerase I is fixed within the nucleus of HeLa cells. EMBO J. 9,2207 -2214.[Abstract]
Felsenfeld, G. (1996). Chromatin unfolds. Cell 86,13 -19.[Medline]
Francastel, C., Walters, M. C., Groudine, M. and Martin, D. I. K. (1999). A functional enhancer supresses silencing of a transgene and prevents its localization close to centromeric heterochromatin. Cell 99,259 -269.[Medline]
Gasser, S. M., Gotta, M., Renauld, H., Laroche, T. and Cockell, M. (1998). Nuclear organization and silencing: trafficking of Sir proteins. CIBA F Symp. 214,114 -126.
Iborra, F. J., Pombo, A., Jackson, D. A. and Cook, P. R.
(1996). Active RNA polymerases are localized within discrete
transcription factories in human nuclei. J. Cell Sci.
109,1427
-1436.
Jackson, D. A., Hassan, A. B., Errington, R. J. and Cook, P. R. (1993) Visualization of focal sites of transcription within human nuclei. EMBO J. 12,1059 -1065.[Abstract]
Jeddeloh, J. A., Stokes, T. L. and Richards, E. J. (1999). Maintenance of genomic methylation requires a SWI2/SNF2-like protein. Nat. Genet. 22, 94-97.[CrossRef][Medline]
Jefferson, R. A., Kavanagh, T. A. and Bevan, M. W. (1987). GUS fusions: ß-glucoronidase as a sensitive and versatile gene fusion marker in plants. EMBO J. 6,3901 -3907.[Abstract]
Jones, P. L., Veenstra, G. J. C., Wade, P. A., Vermaak, D., Kass, S. U., Landsberger, N., Strouboulis, J. and Wolffe, A. P. (1998). Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription. Nat. Genet. 19,187 -191.[CrossRef][Medline]
Kokalj-Vokac, N., Almeida, A., Viegas-Pequignot, E., Jeanpierre, M., Malfoy, B. and Dutrillaux, B. (1993). Specific induction of uncoiling and recombination by azacytidine in classical satellite-containing constitutive heterochromatin. Cytogenet. Cell Genet. 63,11 -15.[Medline]
Kouzarides, A. (2000). Acetylation: a
regulatory modification to rival phosphorylation? EMBO
J. 19,1176
-1179.
Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227,680 -685.[Medline]
Laroche, T., Martin, S. G., Tsai-Pflugfelder, M. and Gasser, S. M. (2000) The dynamics of yeast telomeres and silencing proteins through the cell cycle. J. Struct. Biol. 129,1047 -1077.
Loidl, P. (1994). Histone acetylation: facts and questions. Chromosoma 103,441 -449.[CrossRef][Medline]
Lundgren, M., Chow, C.-M., Sabbattini, P., Georgiou, A., Minaee, S. and Dillon, N. (2000). Transcription factor dosage affects changes in higher order structure associated with activation of a heterochromatic gene. Cell 103,733 -743.[Medline]
Meehan, R. R., Lewis, J. D., Mckay, S., Kleiner, E. L. and Bird, A. P. (1989). Identification of a mammalian protein that binds specifically to DNA containing methylated CpGs, Cell 58,499 -507.[Medline]
Meehan, R. R., Lewis, J. D. and Bird, A. P. (1992). Characterization of MeCP2, a vertebrate DNA binding protein with affinity for methylated DNA. Nucleic Acids Res. 20,5085 -5092.[Abstract]
Nan, X., Tate, P., Li, E. and Bird, A. P. (1996). MeCP2 is a transcriptional repressor with abundant binding sites in genomic chromatin. Mol. Cell. Biol. 16,414 -421.[Abstract]
Nan, X. S., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N. and Bird, A. (1998). Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex. Nature 393,386 -389.[CrossRef][Medline]
Neves, N., Heslop-Harrison, J. S. and Viegas, W. (1995) rRNA gene activity and control of expression mediated by methylayion and imprinting during embryo development in wheat x rye hybrids. Theor. Appl. Genet. 91,529 -533.
Ng, H.-H. and Bird, A. (1999) DNA methylation and chromatin modification. Curr. Opin. Genet. Dev. 9, 158-163.[CrossRef][Medline]
Panstruga, R., Buschges, R., Piffanelli, P. and Schulze-Lefert,
P. (1998). A contiguous 60 Kb genomic stretch from barley
reveals molecular evidence for gene islands in a monocot genome.
Nucleic Acids Res. 26,1056
-1062.
Schwarzacher, T., Heslop-Harrison, J. S., Anamthawat-Jónsson, K. and Finch, R. A. (1992). Parental genome separation in reconstructions of somatic and premeiotic metaphases of Hordeum vulgarex H. bulbosum. J. Cell Sci. 101,13 -24.
The Arabidopsis Genome Initiative (2000). Analysis of the genome sequence of the flowering plant Arabidopsis thaliana. Nature 408,796 -815.[CrossRef][Medline]
Thompson, W. F., Beven, A., Wells, B. and Shaw, P. (1997). Sites of rDNA transcription are widely dispersed through the nucleolus in Pisum sativum and can comprise single genes. Plant J. 12,571 -581.[CrossRef][Medline]
Towbin, H., Staehlin, T. and Gordon, J. (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: Procedure and some applications. Proc. Natl. Acad. Sci. USA 76,4350 -4354.[Abstract]
Travers, A. (1999). An engine for nucleosome remodeling. Cell 96,311 -314.[Medline]
Tumbar, T. and Belmont, A. S. (2001). Interphase movements of a DNA chromosome region modulated by VP16 transcriptional activator. Nat. Cell Biol. 3, 134-139.[CrossRef][Medline]
Vischer, N. O., Huls, P. G. and Woldringh, C. L. (1994). Object-Image: an interactive image analysis program using structured point collection. Binary 6, 35-41.
Wells, B. (1985). Low temperature box and tissue handling device for embedding biological tissue for immunostaining in electron microscopy. Micron. Microsc. Acta 16, 49-53.
Wolffe, A. P. and Guschin, D. (2000). Chromatin structural features and targets that regulate transcription. J. Struct. Biol. 129,102 -122.[CrossRef][Medline]
Wolffe, A. P. and Matzke, M. A. (1999)
Epigenetics: regulation through repression. Science
286,481
-486.
Workman, J. L. and Kingston, R. E. (1998). Alteration of nucleosome structure as a mechanism of transcriptional regulation. Annu. Rev. Biochem. 67,545 -579.[CrossRef][Medline]
Yoshida, M. and Beppu, T. (1988) Reversible arrest of proliferation of rat 3Y1 fibroblasts in both the G1 and G2 phases by trichostatin A. Exp. Cell Res. 177,122 -131.[Medline]
Yoshida, M., Kijima, M., Akita, M. and Beppu, T.
(1990) Potent and specific inhibition of mammalian histone
deacetylase both in vivo and in vitro by trichostatin A. J. Biol.
Chem. 265,17174
-17179.