Tandem repetitive transgenes and fluorescent chromatin tags alter local interphase chromosome arrangement in Arabidopsis thaliana.
ABSTRACT Fluorescent protein chromatin tagging as achieved by the lac operator/lac repressor system is useful to trace distinct chromatin domains in living eukaryotic nuclei. To interpret the data correctly, it is important to recognize influences of the tagging system on nuclear architecture of the host cells. Within an Arabidopsis line that carries lac operator/lac repressor/GFP transgenes, the transgene loci frequently associate with each other and with heterochromatic chromocenters. Accumulation of tagged fusion protein further enhances the association frequency. Independent experiments with a transgenic plant carrying another multi-copy transgene also revealed, independent of its transcriptional state, unusually high frequencies of association with each other and with heterochromatin. From these results we conclude that the lac operator/lac repressor chromatin tagging system may alter the spatial chromatin organization in the host nuclei (in particular when more than one insertion locus is present) and also that loci of homologous transgenic repeats associate more often with each other and with endogenous heterochromatin than normal euchromatic regions.
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ABSTRACT: Chromosomally-integrated arrays of lacO and tetO operator sites visualized by LacI and TetR repressor proteins fused with GFP (or other fluorescent proteins) are widely used to monitor the behavior of chromosomal loci in various systems. Yet, insertion of such arrays and expression of the corresponding proteins is known to perturb genomic architecture. In several cases, juxtaposition of such arrays located on different chromosomes has been inferred to reflect pairing of the corresponding loci. Here, we report that a version of TetR-GFP mutated to disrupt GFP dimerization (TetR-A206KGFP or "TetR-kGFP"), abolishes pairing of tetO-arrays in vivo and brings spatial proximity of chromosomal loci marked with those arrays back to the wild type level. These data argue that pairing of arrays is caused by GFP dimerization and thus presents an example of protein-assisted interaction in chromosomes. Arrays marked with another protein, TetR-tdTomato, which has a propensity to form intra-molecular dimers instead of inter-molecular dimers, also display reduced level of pairing, supporting this idea. TetR-kGFP provides an improved system for studying chromosomal loci with a low pairing background.Journal of Molecular Biology 11/2013; · 3.91 Impact Factor
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ABSTRACT: Paramutation is a well-studied epigenetic phenomenon in which trans communication between two different alleles leads to meiotically heritable transcriptional silencing of one of the alleles. Paramutation at the b1 locus involves RNA-mediated transcriptional silencing and requires specific tandem repeats that generate siRNAs. This study addressed three important questions: 1) are the tandem repeats sufficient for paramutation, 2) do they need to be in an allelic position to mediate paramutation, and 3) is there an association between the ability to mediate paramutation and repeat DNA methylation levels? Paramutation was achieved using multiple transgenes containing the b1 tandem repeats, including events with tandem repeats of only one half of the repeat unit (413 bp), demonstrating that these sequences are sufficient for paramutation and an allelic position is not required for the repeats to communicate. Furthermore, the transgenic tandem repeats increased the expression of a reporter gene in maize, demonstrating the repeats contain transcriptional regulatory sequences. Transgene-mediated paramutation required the mediator of paramutation1 gene, which is necessary for endogenous paramutation, suggesting endogenous and transgene-mediated paramutation both require an RNA-mediated transcriptional silencing pathway. While all tested repeat transgenes produced small interfering RNAs (siRNAs), not all transgenes induced paramutation suggesting that, as with endogenous alleles, siRNA production is not sufficient for paramutation. The repeat transgene-induced silencing was less efficiently transmitted than silencing induced by the repeats of endogenous b1 alleles, which is always 100% efficient. The variability in the strength of the repeat transgene-induced silencing enabled testing whether the extent of DNA methylation within the repeats correlated with differences in efficiency of paramutation. Transgene-induced paramutation does not require extensive DNA methylation within the transgene. However, increased DNA methylation within the endogenous b1 repeats after transgene-induced paramutation was associated with stronger silencing of the endogenous allele.PLoS Genetics 10/2013; 9(10):e1003773. · 8.52 Impact Factor
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ABSTRACT: Plant genomes are extremely sensitive to, and can be developmentally reprogrammed by environmental light cues. Here using rolling-circle amplification of gene-specific circularizable oligonucleotides coupled with fluorescence in situ hybridization, we demonstrate that light triggers a rapid repositioning of the Arabidopsis light-inducible chlorophyll a/b-binding proteins (CAB) locus from the nuclear interior to the nuclear periphery during its transcriptional activation. CAB repositioning is mediated by the red/far-red photoreceptors phytochromes (PHYs) and is inhibited by repressors of PHY signalling, including COP1, DET1 and PIFs. CAB repositioning appears to be a separate regulatory step occurring before its full transcriptional activation. Moreover, the light-inducible loci RBCS, PC and GUN5 undergo similar repositioning behaviour during their transcriptional activation. Our study supports a light-dependent gene regulatory mechanism in which PHYs activate light-inducible loci by relocating them to the nuclear periphery; it also provides evidence for the biological importance of gene positioning in the plant kingdom.Nature Communications 01/2014; 5:3027. · 10.02 Impact Factor
In situ localization and direct in vivo visualization of distinct
chromosome regions recently became feasible by chromatin-
tagging systems. The lac operator/lac repressor system
(Robinett et al., 1996; Straight et al., 1996) for instance uses a
bacterial DNA binding protein (lac repressor) that, when fused
with a green fluorescent protein (GFP) and a nuclear
localization signal peptide (NLS), binds to the 256 copies of
directly repeated lac operators (~10 kb) integrated at a specific
chromosome locus. Binding at the target locus yields a
fluorescent spot of higher intensity than the overall
fluorescence of dispersed unbound GFP molecules in the
nucleoplasm. The GFP-lac repressor protein is either
transiently or stably expressed. The system was applied to
various eukaryotes such as yeasts (Aragon-Alcaide and
Strunnikov, 2000; Nabeshima et al., 1998; Straight et al.,
1996), flies (Gunawardena and Rykowski, 2000; Vazquez et al.,
2002), cultured mammalian cells (Robinett et al., 1996;
Tsukamoto et al., 2000) and plants (Esch et al., 2003; Kato
and Lam, 2001; Matzke et al., 2003). It revealed structural
dynamics of chromosomes in interphase as well as mitotic cells
by tracing the tagged loci in living cells and contributed to the
understanding of chromosome functions (reviewed by Belmont
et al., 1999; Gasser, 2002; Lam et al., 2004). However, tagging
systems represent artificial chromosome loci and sometimes
generate unusual nuclear protein localization. For instance in
baby hamster kidney cells in which a lac operator array is
amplified about ten times, a nuclear protein complex, the PML
(promyelocytic leukemia) body, is formed at the integration
locus (Tsukamoto et al., 2000). PML bodies are thought to play
a role in regulating transcription. Because PML bodies are not
formed at the transgene locus without accumulation of the lac
operator binding fusion protein, they are thought to recognize
high concentrations of induced foreign proteins (Tsukamoto et
Previously, we used the lac operator/lac repressor system to
compare chromosome dynamics in Arabidopsis nuclei of
different ploidy level (Kato and Lam, 2003), for example in 2C
nuclei of guard cells (stomata) and in nuclei of pavement cells
(8C on average). We found that chromosomes in interphase
nuclei of Arabidopsis move randomly within a restricted area
and that the area size correlates with the nuclear DNA content.
Lac operator loci seemed to be stochastically associated in
diploid as well as in endopolyploid nuclei according to the GFP
spot numbers observed (Kato and Lam, 2003). Contrary to this,
Esch and colleagues (Esch et al., 2003) reported that tagged
lac operator loci in Arabidopsis are tightly, but not
stochastically associated with each other because only one GFP
spot was observed in most diploid as well as in polyploid cells.
Therefore it was necessary to address the question of whether
Fluorescent protein chromatin tagging as achieved by the
lac operator/lac repressor system is useful to trace distinct
chromatin domains in living eukaryotic nuclei. To interpret
the data correctly, it is important to recognize influences of
the tagging system on nuclear architecture of the host cells.
Within an Arabidopsis line that carries lac operator/lac
repressor/GFP transgenes, the transgene loci frequently
associate with each other and with heterochromatic
chromocenters. Accumulation of tagged fusion protein
further enhances the association frequency. Independent
experiments with a transgenic plant carrying another
multi-copy transgene also revealed, independent of its
transcriptional state, unusually high frequencies of
association with each other and with heterochromatin.
From these results we conclude that the lac operator/lac
repressor chromatin tagging system may alter the spatial
chromatin organization in the host nuclei (in particular
when more than one insertion locus is present) and also that
loci of homologous transgenic repeats associate more often
with each other and with endogenous heterochromatin
than normal euchromatic regions.
Key words: Arabidopsis thaliana, Fluorescent chromatin tag,
Homologous pairing, Interphase chromosomes, Heterochromatin
Tandem repetitive transgenes and fluorescent
chromatin tags alter local interphase chromosome
arrangement in Arabidopsis thaliana
Ales Pecinka1,‡, Naohiro Kato2,*,‡, Armin Meister1, Aline V. Probst3, Ingo Schubert1and Eric Lam2,§
1Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany
2Biotech Center for Agriculture and the Environment, Rutgers, The State University of New Jersey, 59 Dudley Road, Foran Hall, New Brunswick,
NJ 08901, USA
3Laboratory of Plant Genetics, University of Geneva, 30 Quai Ernset Ansermet, CH-1211 Geneva 4, Switzerland
*Present address: Department of Biological Sciences, Louisiana State University, 226 Life Science Buildings, Baton Rouge, LA 70808, USA
‡These authors contributed equally to this work
§Author for correspondence (e-mail: firstname.lastname@example.org)
Accepted 13 May 2005
Journal of Cell Science 118, 3751-3758 Published by The Company of Biologists 2005
Journal of Cell Science
lower-than-expected numbers of GFP spots are indeed due to
associations of homologous loci in Arabidopsis nuclei and, if
so, to determine why this is occurring.
Here we determined the number of lac operator FISH
(fluorescent in situ hybridization) signals before and after
expression of the GFP-lac repressor protein and the proportion
of loci exhibiting a GFP spot after induced expression.
Furthermore we compared the spatial organization of the
tagged chromatin in hemizygous and homozygous conditions.
The aim was to test whether the repeat structure and/or the
expression of the GFP-lac repressor protein of the transgene
have an impact on allelic/ectopic homologous pairing and on
association with heterochromatic domains of the regions in
question in 2C leaf nuclei. We found that the lac operator
arrays are associated with each other more often than predicted
by computer model simulation (Pecinka et al., 2004).
Expression of the GFP-lac repressor protein further increases
the homologous association frequency. Because the
chromosome regions adjacent to the tagged loci associated
with each other as well as with heterochromatin less often than
lac operator arrays, we conclude that homologous tandem
repetitive transgenes preferentially associate with each other as
well as with endogenous heterochromatin in A. thaliana. In
independent experiments homologous pairing and association
with heterochromatin were studied for a homozygous multi-
copy hygromycin phosphotransferase (HPT) transgene locus of
~100 kb (Probst et al., 2003) and yielded similar results to
those obtained for the homozygous lac operator loci.
Materials and Methods
Plant material, preparation of nuclei and pachytene
Plants of A. thaliana accession Columbia (Col) of hemizygous and
homozygous EL702C genotypes (Kato and Lam, 2003), homozygous
EL700S genotype (Kato and Lam, 2001; Kato and Lam, 2003) or
homozygous line A and the mutant mom1-1 genotype that contains
the HPT locus homozygously (Probst et al., 2003) were cultivated as
described. To induce expression of the GFP-lac repressor protein,
young rosette leaves were detached from the plants and floated on 10
ml of 0.3 μM (homozygous EL702C) or 3 μM (wild type,
homozygous EL700S) dexamethasone (Dex) solution in Petri dishes
for 6-12 hours. After fixation of leaves in 4% paraformaldehyde, 2C
nuclei were flow-sorted as described (Jasencakova et al., 2000). Prior
to FISH, Dex-treated 2C nuclei were analyzed for the presence of GFP
spots and the positions on slides of nuclei with GFP spots were
recorded using a microscopic grid slide. Pachytene chromosomes of
the wild type and homozygous EL702C genotypes were prepared as
described (Lysak et al., 2001).
Fluorescence microscopy for GFP spot detection in living
guard cell nuclei of Arabidopsis seedlings
The GFP signals were detected as described (Kato and Lam, 2003).
Spots were defined as pixel clusters comprising more than the mean
number of pixels plus 3.3-fold the value of the s.d. in the analyzed
Probe labeling and in situ hybridization
The following DNA clones were used as probes: BAC MGL6
(GenBank accession number AB022217), BAC F18C1 (AC011620),
BAC T15P10 containing 45S rDNA sequence (AF167571), 128x
lacO-SK (Kato and Lam, 2001), pAL1 (Martinez-Zapater et al.,
1986), the BAC contig spanning 4.2 Mb of chromosome 3 top arm
from F2O10 to MSL1 (AC013454 and AB012247, respectively) and
pGL2 (containing the hygromycin phosphotransferase gene under the
control of the CaMV 35S promoter). BAC and plasmid DNA was
labeled by nick translation (Ward, 2002) or by PCR using biotin-2’-
deoxyuridine 5′-triphosphate (biotin-dUTP) (Probst et al., 2003). For
chromosome painting, labeled BACs were precipitated and
resuspended in 20 μl hybridization buffer (50% formamide, 10%
dextran sulphate, 2? SSC, 50 mM sodium phosphate, pH 7.0) per
slide. After mounting the probe, the slides were placed on a heat block
at 80°C for 2 minutes and then incubated in a moist chamber at 37°C
for ~12 hours. Post hybridization washes and detection steps were
conducted as described (Schubert et al., 2001). Biotin-dUTP was
detected by avidin conjugated with Texas Red (1:1000; Vector
Laboratories), biotin-conjugated goat anti-avidin (1:200; Vector
Laboratories), and again with Texas Red-conjugated avidin.
Digoxigenin-dUTP was detected by mouse anti-digoxigenin (1:250;
Roche) and Alexa 488-conjugated goat anti-mouse antibody (1:200;
Molecular Probes). Cy3-dUTP was observed directly. Nuclei and
chromosomes were counterstained with 1 μg/ml DAPI in Vectashield
mounting medium (Vector Laboratories).
Microscopic evaluation and image processing
Fluorescence signals in flow-sorted 2C nuclei and on pachytene
chromosomes were analyzed using an Axioplan 2 (Zeiss)
epifluorescence microscope with 100?/1.4 Zeiss plan apochromat
objective. Images were acquired with MetaVue (Universal Imaging)
software and a cooled charge-coupled device camera (Spot 2e,
Diagnostic Instruments) separately for each fluorochrome using the
appropriate excitation and emission filters. The monochromatic
images were pseudocolored, Gauss- or median-filtered to reduce noise
and merged using Adobe Photoshop 6.0 (Adobe Systems) software.
A spatial overlap of compact spheric FISH signals of homologous
and/or heterologous sequences was regarded as homologous pairing
and heterologous association, respectively. Allelic versus ectopic
pairing of transgenic loci was distinguished on the basis of FISH
signals obtained from differently labeled BACs that contain sequences
flanking the respective transgene insertion loci.
GFP spot numbers vary in 2C nuclei of live transgenic
In our previous studies, we rarely observed four GFP spots in
2C guard cell nuclei in homozygous EL702C plants with two
loci on chromosome 3 containing lac operator insertions (Kato
and Lam, 2003). Here we compare spot numbers in guard cell
nuclei of cotyledons of hemizygous and homozygous EL702C
plants and of homozygous transgenic EL700S plants (Fig. 1).
As EL700S plants contain the same construct as EL702C
plants except for the lac operator array, homogeneously
distributed GFP signals but no GFP spots were expected in the
nucleoplasm. In hemizygous EL702C plants, we found 5% of
92 nuclei without spots, 66% with one spot, 27% with two and
2% with three spots. In homozygous EL702C plants 12% of
197 nuclei had no spots, 34% showed one spot, 37% two, 11%
three and 6% four spots. In homozygous EL700S plants, 55
out of 56 nuclei showed no spots and one nucleus (<2%)
showed one spot. The single spot observed in an EL700S
nucleus and a third spot in two hemizygous EL702C nuclei are
most likely caused by spontaneous aggregation of GFP-lac
repressor-NLS molecules. Taking into account our not being
able to observe the maximum number of GFP spots in all nuclei
Journal of Cell Science 118 (16)
Journal of Cell Science
Interaction between inserted repeat arrays
and in order to understand the nature of GFP-spot appearance
patterns in the nuclei, we compared the observed data with the
statistically expected percentages for nuclei with different spot
numbers. We considered two statistical models.
In the first model, we assume that all lac operator loci in a
nucleus appear as a GFP spot at random and that the two loci
in the EL702C insertion line have an equal probability of
interaction with GFP-lac repressor-NLS fusion proteins. In this
model, spot detection should follow a binomial distribution
with an average probability of spot appearance p estimated as,
p = (0?n0+ 1?n1 + … + N?nN)/(n0+ n1 + … + nN)/N,
where N is the maximum number of spots per nucleus (N=2
for hemizygous, N=4 for homozygous nuclei) and niis the
number of nuclei with i spots (i=0 … N). The probability of
spot appearance calculated from the experimental data is
significantly different for hemizygous (P=0.61) and for
homozygous (P=0.41) nuclei (χ2test, P<0.001). Moreover, the
distribution of the observed frequencies for different spot
numbers per nucleus deviate significantly from the binomial
distribution assuming the corresponding probability (χ2=15.20,
df=1, P=0.0001 for hemizygous nuclei; χ2=10.46, df=3,
P=0.015 for homozygous nuclei).
We previously observed that lac operator repeats with
different lengths of the array may be detected as GFP spots with
different probabilities (Kato and Lam, 2001). Therefore, in the
second model we assumed that GFP spots appear with different
probabilities on each tagged locus, possibly due to the different
sizes of lac operator array in these loci (a single array in the
distal and two lac operator arrays in the proximal insertion locus
in the top arm of chromosome 3 of the EL702C line). We first
estimated the probability values p1and p2for the two loci in
hemizygous nuclei. On the basis of the observed percentages of
hemizygous nuclei with one spot: p1(1–p2) + p2(1–p1) =0.66 or
with two spots: p1·p2=0.27, we calculated: p1=0.30 and p2=0.93.
For homozygous nuclei, p1=0.24 and p2=0.55 were calculated
similarly by numerical solution of the corresponding equations.
With these estimated probabilities, the observed frequencies of
hemizygous nuclei with 0, 1 or 2 spots and of homozygous
nuclei with 0, 1, 2, 3 or 4 spots fitted very well with the expected
values. However, our result suggests that the probability of spot
appearance in hemizygous nuclei is higher than those of
homozygous nuclei. Although non-homogeneous GFP
accumulation in a nucleus or different levels of accumulation in
hemizygous compared to homozygous nuclei might explain the
lower probability of GFP spot appearance in homozygous
nuclei, we preferred the hypothesis that the tagged loci might
have intrachromosomal and/or interchromosomal interactions
in the nuclei of Arabidopsis, thereby altering the apparent spot
frequencies. To test this hypothesis, we analyzed the positional
coincidence of the integrated lac operator loci in isolated 2C
leaf nuclei by FISH analyses.
GFP spots and FISH signals of lac operator arrays
frequently colocalize in homozygous EL702C nuclei
In flow-sorted 2C nuclei of homozygous EL702C plants in
which expression of the GFP-lac repressor protein was induced
with Dex, GFP spots and FISH signals on the lac operator loci
were counted (Fig. 2). Nuclei without clear GFP spots were
excluded from evaluation. Out of 63 analyzed nuclei, 30%
showed one, 35% two, 25% three and 10% four GFP spots. In
contrast, 22% of nuclei showed four FISH signals, 35%
showed two or three signals and 8% showed one FISH signal.
All GFP spots coincided with a lac operator FISH signal (Fig.
2B) but not vice versa. In total 83% of 252 FISH signals
coincided with a GFP signal. Thus, 17% of the transgene loci
cannot be detected by a GFP spot in Dex-treated homozygous
EL702C nuclei under the applied conditions. Either not all lac
operator arrays were accessible to the GFP-lac repressor
proteins or some GFP spots could not be discriminated owing
to a high overall fluorescence intensity and/or rapid bleaching
of signals within a minute of exposure. We hypothesized that
less than four FISH signals per nucleus may be due to ectopic
or allelic alignment of the lac operator arrays.
The two transgene loci of EL702C are separated by a
pericentrically inverted region
The transgenic line EL702C carries three transgenes (each ~17
kb) inserted at two independent loci on the top arm of
chromosome 3, ~4.2 Mb apart; the proximal locus harbors two
transgenes in inverse orientation (Kato and Lam, 2003). FISH
analyses with BAC probes containing insert sequences
internally flanking the transgene loci were used to quantify
ectopic and allelic pairing frequencies of the lac operator
arrays in 2C nuclei. To confirm the physical position of the
transgene and BAC loci on chromosome 3, we first hybridized
these probes to pachytene chromosomes of EL702C. The lac
operator probes hybridized to the predicted locations whereas
Fig. 1. GFP spot numbers in living guard cell nuclei (2C) from
cotyledons of dexamethasone-treated transgenic Arabidopsis
seedlings. (A) Percentage of nuclei with 0, 1, 2, 3 and 4 spots in
hemizygous EL702C plants (n=92), homozygous EL702C plants
(n=197) and homozygous EL700S plants (n=56). (B) Representative
images of nuclei with 0, 1, 2, 3 and 4 spots from each of the lines and
schematic view of the lac operator array loci on chromosome 3 in
Journal of Cell Science
the BAC probes hybridized in reverse order suggesting an
inversion of the region between the transgene loci. Also FISH
with two differently labeled BAC pools (MBK21 to MSL1
corresponding to the upper region and F2O10 to F28J15
corresponding to the bottom region between the insertion
loci) yielded signals of reversed orientation on pachytene
chromosomes of homozygous EL702C compared to that in
wild-type plants (Fig. 3). FISH signals of the BACs flanking
the insertion loci externally appeared in the same order on the
wild-type and EL702C bivalents (not shown). These results
confirmed the inversion between the transgene loci on the top
arm of chromosome 3 in EL702C plants.
Ectopic pairing of lac operator arrays is doubled in
homozygous nuclei compared to hemizygous nuclei
Using tri-color FISH with BAC MGL6 (79.5 kb, ~54 kb
downstream of the insertion, red) flanking the distal locus,
BAC F18C1 (100.8 kb, ~55 kb upstream of the insertion,
yellow) flanking the proximal locus and lac operator probe
(green) (Fig. 4B-D), the ectopic and allelic pairing frequency
of the lac operator arrays was assessed in 60 hemizygous
untreated, 62 homozygous untreated and 59 homozygous Dex-
treated EL702C nuclei. We classified the lac operator array
alignments into two different pairing types. If two signals
(MGL6, red and F18C1, yellow) colocalized with a lac
operator signal (green), we identified the alignment as ectopic
pairing. If all signals of either MGL6 or F18C were colocalized
with a lac operator signal, we identified the alignment as allelic
pairing. In hemizygous nuclei, ectopic pairing was detected for
13% of the lac operator loci without Dex treatment. In
homozygous EL702C nuclei without Dex treatment, ectopic
pairing was observed for 27% of the lac operator array loci and
allelic pairing for 34% of the loci. With Dex treatment, these
values increased to 35% (ectopic pairing, P=0.052) and 45%
(allelic pairing, P=0.017), respectively.
Homologous pairing and ectopic association of regions
flanking the transgene are more frequent in EL702C
than in wild-type plants
Using the BACs F18C1 (yellow) and MGL6 (red) that flank
the transgene insertions in EL702C nuclei as probes, two-color
FISH was conducted to monitor the association of these
regions in wild-type nuclei (n=153), hemizygous (n=60) and
homozygous (n=62) EL702C nuclei without Dex treatment.
We also analyzed 61 wild-type nuclei and 59 homozygous
EL702C nuclei after Dex treatment (Fig. 5). Homologous
pairing of both regions as well as their heterologous association
occurred without significant differences (i.e. 3-6% per locus,
see Fig. 5A) in wild-type nuclei and hemizygous EL702C
nuclei, irrespective of Dex treatment. In homozygous EL702C
nuclei, homologous pairing (10%, P<0.05 for MGL6 and 14%,
P<0.001 for F18C1) and ectopic association (9%, P>0.05)
occurred more often than in the wild type. A further increase
Journal of Cell Science 118 (16)
Fig. 2. Colocalization of GFP spots and lac operator FISH signals in
Dex-treated 2C leaf nuclei of homozygous EL702L plants.
(A) Percentage of nuclei with one to four GFP spots compared to
FISH signals. Fewer GFP spots than FISH signals may occur in one
nucleus. (B) Examples of nuclei with 1, 2, 3 or 4 GFP spots
coinciding with lac operator FISH signals. Bars, 3 μm.
Fig. 3. The double transgene insertion in EL702C is accompanied by
a paracentric inversion between the integration points. (A) Painted
regions between BACs F2O10 and F28J15 (yellow) and MBK21 and
MSL1 (red) schematically positioned on the top arm of chromosome
3 of the wild-type (left) and together with the transgene (green) of
the EL702C genotype (right). (B) FISH of the complex probe to
pachytene chromosomes of the wild type (left) and homozygous
EL702C (right). Arrows indicate the top arm end of chromosome 3
bivalent. Bars, 5 μm.
Journal of Cell Science
Interaction between inserted repeat arrays
of homologous pairing (20%, P=0.032; and 22%, P=0.177,
respectively) as well as of ectopic association (17%, P=0.013;
Fig. 5A) was found after induction of GFP-lac repressor
protein expression in homozygous EL702C nuclei (all at
P<0.001 when compared to levels in the wild type).
The transgene colocalizes more often than the flanking
regions with heterochromatic chromocenters
During the FISH analysis described above, we noticed a
frequent spatial association of lac operator loci with
heterochromatic chromocenters that are detected as strongly
DAPI-stained regions. Therefore we determined the frequency
of positional overlap (colocalization) of FISH signals of
F18C1, MGL6 and lac operator probes with strongly DAPI-
stained chromocenters in homozygous EL702C nuclei without
(n=41) and with (n=31) Dex treatment. For comparison, the
overlap of FISH signals of MGL6 and F18C1 probes with those
of centromeric repeats and 45S rDNA, the main components
of heterochromatin in Arabidopsis (Fransz et al., 2002), were
monitored in 62 wild-type nuclei (Fig. 6). Although 8-14%
of MGL6 and F18C1 FISH signals colocalized with
heterochromatin in all types of nuclei tested, 37% of lac
operator signals overlapped with chromocenters in untreated
and 44% in Dex-treated homozygous EL702C nuclei (both
P<0.001 when compared to the flanking regions). Apparently,
the colocalization of lac operators with heterochromatin did
not interfere with expression of the GFP-lac repressor protein
in homozygous EL702C nuclei although the lac repressor gene
is situated close to the lac operator array (Fig. 6). In order to
test whether pairing of lac operator loci precedes association
with heterochromatin, we counted the number of lac operator
loci per overlap with a chromocenter. Within 31 Dex-treated
homozygous EL702C nuclei (harboring 124 loci), 54 loci
associated with a chromocenter, of which 14 were detected as
a single locus, 24 as two, 12 as three and 4 as four paired loci
suggesting that transgene pairing is not a prerequisite for
association with heterochromatin.
Fig. 4. Ectopic and allelic pairing of the lac operator array loci in
EL702C plants. (A) Percentage of loci showing ectopic pairing
(untreated hemizygous nuclei) or ectopic/allelic pairing (untreated
and Dex-treated homozygous nuclei). (B) Scheme of chromosome 3
(EL702C) with the transgene insertion positions and the flanking
regions used as markers for FISH. (C) Hemizygous nuclei showing
ectopic pairing (top) or separation (bottom) of transgenic loci.
(D) Homozygous nuclei with ectopic pairing of both transgenic loci
(top, could be intrachromosomally or between homologues), allelic
pairing of only the distal locus (middle) or separation of both loci
(bottom). Bars, 3 μm.
Fig. 5. Homologous pairing and ectopic association of regions
flanking the lac operator transgene. (A) Percentage of homologously
paired MGL6 loci, homologously paired F18C1 loci and association
between both regions in the wild type, hemizygous and homozygous
EL702C nuclei without or after Dex treatment. Significant
differences *P<0.05 and ***P<0.001 were found compared to levels
in the wild type determined using χ2test. (B) Schemes of
chromosome 3 (wild type and EL702C) showing the position of
BACs MGL6 and F18C1 used for FISH. (C) Homozygous EL702C
nuclei showing homologous pairing of MGL6 (top) or ectopic
association intrachromosomally or between two homologues
(bottom). Bars, 3 μm.
Journal of Cell Science
Pairing behavior and association frequency with
heterochromatin of the lac operator array may not be
To test whether or not the higher-than-random allelic pairing
frequency of the lacoperator array is true only for this sequence,
the pairing frequency of the homozygous silent transgenic HPT
locus (composed of ~15 rearranged plasmid copies of ~100 kb
in A. thaliana line A) (Mittelsten Scheid et al., 1991; Mittelsten
Scheid et al., 1998) was investigated by FISH (Fig. 7). For
comparison, the same locus was tested in a mom1-1 mutant
background (Amedeo et al., 2000) where HPT silencing is
released without alteration of DNA methylation and histone
modifications (Probst et al., 2003). In nuclei of line A, 30% of
HPT FISH signals were paired. This value is significantly
higher (P<0.001) than the ~5% of pairing observed on average
for FISH signals of BACs with inserts from various endogenous
euchromatic regions along the Arabidopsis chromosomes
(Pecinka et al., 2004) but not significantly different (P>0.05)
from the allelic pairing frequency of transgenic lac operator
arrays (34% of loci) in homozygous EL702C nuclei. In mom1-
1 nuclei where HPT genes are expressed, association of HPT
FISH signals (21%) was still significantly higher than the
average pairing frequency (P<0.001). Colocalization with
heterochromatic chromocenters was found for 50% of HPT
signals in line A and for 49% in mom1-1 nuclei. In 269 line A
nuclei, 165 out of the 271 HPT loci colocalizing with
chromocenters associated as a single locus and 106 as paired
loci. In 355 mom1-1 nuclei, 224 out of 346 loci colocalizing
with a chromocenter associated as single and 122 as paired loci.
Because 60-65% of heterochromatin-associated loci were not
paired, homologous pairing seems not to be a prerequisite for
spatial association of HPT loci with chromocenters. Hence the
association frequency of the HPT locus with heterochromatin
is even higher than that observed for the lac operator arrays,
independent of the transcriptional status and of previous
FISH analyses of the transgenic line EL702C with flanking
BACs revealed a previously undetected inversion between the
two insertion loci of lac operator arrays on the top arm of
chromosome 3. Without sequencing the actual breakpoints
(~10-55 kb away from the insertions) we are currently not able
to identify the molecular event responsible for that inversion
and to decide on one of the models proposed for the generation
of inversions during insertion of two transgenes in cis
configuration (Laufs et al., 1999).
Homologous pairing of ~100 kb regions along different
chromosomes of A. thaliana accession Columbia occurs on
average in about 5% of somatic nuclei (Pecinka et al., 2004).
In wild-type nuclei, allelic pairing and ectopic association of
the regions that flank the lac operator loci in EL702C occur
with a similar frequency (3-6% per locus). These values are
within the range predicted for random appearance of ‘single
point’ homologous pairing according to simulations based on
the ‘random spatial distribution’ model (Pecinka et al., 2004).
The homozygous presence of the lac operator arrays results in
a four- to tenfold higher frequency of allelic as well as of
ectopic pairing of these loci compared to the average values
observed for endogenous sequences in wild-type nuclei
(compare values for flanking sequences in Fig. 5A with those
for lac operator arrays in Fig. 4A). The high allelic pairing
frequency of the transgene may exert a ‘dragging’ effect on the
flanking regions (Fig. 5A). In hemizygotes, a dragging effect
is not obvious because pairing of the transgene is less frequent
than in homozygotes and in most cases FISH signals of
flanking regions are separated by those of lac operator loci
Journal of Cell Science 118 (16)
Fig. 6. Association frequency of the lac operator arrays with
heterochromatic domains compared to that of flanking MGL6/F18C1
sequences. The percentage of FISH signals associated with
heterochromatin in nuclei of wild type (MGL6, F18C1) and of
homozygous EL702C plants without and after Dex treatment
(MGL6, F18C1, lac operator) is shown. ***P<0.001 compared to the
wild-type situation determined using χ2test.
Fig. 7. The association frequency of the transgenic hygromycin
phosphotransferase (HPT) locus with endogenous heterochromatin is
similar in line A and in the mom1-1 background. Top row,
Arabidopsis nuclei with intense DAPI-stained chromocenters.
Middle row, the same nuclei after FISH with the 178 bp centromeric
repeat (red) and pGL2 sequence labeling the HPT locus (green).
From left to right: two, one or none of the separated allelic HPT
signals associated with centromeric heterochromatin, and paired
HPT signals associated or not associated with centromeric
heterochromatin. Note the appearance of the HPT locus as small
DAPI-positive chromocenter(s) in the second, third and fifth nucleus
of the upper panel (arrows). Bottom row, the percentage of nuclei
showing the corresponding type of association of HPT signals with
centromeric heterochromatin (line A, n=269 nuclei; mom1-1, n=355
nuclei). Of the total HPT signals 30% were paired (see fourth and
fifth nucleus) in line A and 21% in mom1-1. In nuclei of line A, 61%
of the 271 HPT sites colocalizing with centromeric heterochromatin
did so as single sites and 39% as paired sites. In mom1-1 nuclei, 65%
of the 346 HTP sites colocalized with centromeric heterochromatin
as single sites and 35% as paired sites.
Journal of Cell Science
Interaction between inserted repeat arrays
during ectopic transgene pairing. We also found the repeated
HPT locus to be paired significantly more often (21-29%) than
expected according to a random frequency that was observed
for several endogenous euchromatic loci (Pecinka et al., 2004).
From these data we speculate that tandem repetitive sequences
promote homologous association in Arabidopsis. Such a
tendency for homologous association of tandem repeats could
also be the reason for association of multiple transgene
insertion loci in wheat nuclei (Abranches et al., 2000; Santos
et al., 2002). The dispersion of the wheat transgene loci
observed after 5-azacytidine or trichostatin A treatment (Santos
et al., 2002) might be due to chromatin modifications rather
than to transcriptional activity (see below). Expression of the
GFP-lac repressor protein in homozygous EL702C nuclei
yielded a further increase of allelic and ectopic pairing of the
transgene locus by an additional 5-10% (Fig. 4A), with an
additional dragging effect on the flanking regions (Fig. 5A).
Expression of HPT in the mom1-1 background does not
increase homologous pairing of the transgene locus containing
the HPT repeat. We speculate that GFP-lac repressor protein
binding to the lac operator arrays, rather than just expression
of the transgene, enforces allelic and ectopic pairing of the lac
operator arrays. Wild-type lac repressor (tetramerizing form)
can bind lac operators on different DNA molecules, tethering
together loci on different chromosomes (Straight et al., 1996;
Weiss and Simpson, 1997). Because we used a dimerizing
mutant form of the lac repressor (Kato and Lam, 2001), which
can bind only one lac operator site (Robinett et al., 1996), the
capability of tethering two chromosomes should be minimized
in EL702C. Nevertheless, spontaneous association of GFP-lac
repressor protein molecules bound to different lac operator loci
might increase the pairing frequency. Previously, we reported
that movement of tagged chromatin in Arabidopsis nuclei, in
spite of being spatially constrained, may span up to 0.44 μm
within 10 minutes (Kato and Lam, 2003). Because homologous
chromosome regions of ~100 kb are either paired or separated
by less than 0.2 μm in ~20% of Arabidopsis nuclei on average
(Pecinka et al., 2004), it seems reasonable to assume that
during the 12 hours of Dex treatment, random associations of
lac operator sites may occur and they then become stabilized
due to aggregation of GFP-lac repressor proteins.
In contrast to the FISH signals of the lac operator array
flanking regions, of which 8-14% overlapped with
heterochromatin, signals of the inserted lac operator arrays
colocalized as single or paired loci with chromocenters in 37%
of untreated and in 44% of Dex-treated homozygous EL702C
nuclei. As lac operator arrays may associate with
heterochromatin as single or as paired loci, this colocalization
does not depend upon pairing of the repetitive transgene arrays.
Probably, tandem repeat loci tend to associate with each
other on the basis of sequence homology but also with
heterochromatic chromocenters containing other repeat
sequences. This would render tandem repeats better candidates
for anchoring euchromatin loops to heterochromatin according
to the ‘chromocenter-loop model’ (Fransz et al., 2002) than
dispersed repeats such as Emi12 elements that colocalize with
chromocenters only in 1-7% of nuclei (S. Hudakova, IPK,
Gatersleben, Germany and I.S., unpublished results). In total
the HPT locus is clearly larger than the lac operator locus and
often becomes visible as DAPI-intense chromocenter(s) (Fig.
7) (Probst et al., 2003). Because the HPT locus colocalized
more often than the lac operator locus with heterochromatin,
the tendency of tandem repeats to associate with
heterochromatin in Arabidopsis interphase nuclei may
correlate with the size of the entire repeat-containing locus. For
the HPT locus the association with heterochromatin was
independent of transcription. In the case of the lac operator
locus, transcriptional activity of adjacent sequences does not
reduce the frequency of its colocalization with endogenous
heterochromatin. The mechanism by which repeat sequences
are targeted to chromocenters remains to be elucidated.
Finally, our findings suggest that in many nuclei the lac
repressor/lac operator chromatin-tagging system does not
reflect the spatial organization at the integration loci under
wild-type conditions and may lead to invalid conclusions as to
single-point homologous pairing frequencies (Esch et al.,
2003). This problem could become significant especially when
multiple insertions of repetitive arrays are present either in
hemizygous or in homozygous conditions. The main reason for
the increase in allelic and ectopic association frequency of the
lac operator (compared to the flanking sequences in wild-type
conditions) is most likely the repetitive nature of the transgene
construct. Sequence-specific but more or less location-
independent somatic association of multiple inserted arrays of
tet operator and lac operator has been reported for budding
yeast (Aragon-Alcaide and Strunnikov, 2000), although this
was not confirmed by FISH or in the absence of fusion protein.
For the same organism, association of tet operator arrays was
shown to depend on the expression of the tet repressor fusion
protein (Fuchs et al., 2002). In Drosophila, lacO arrays
apparently did not reveal a tendency for homologous pairing
as it was possible to trace extensive separation of homologues
and even of sister chromatids along chromosome arms during
pre-meiotic mid-G2 (Vazquez et al., 2002). Our results
obtained for the HPT locus further support the idea that in A.
thaliana the tandem repetitive nature of a transgene locus
might be responsible for an increased allelic and ectopic
pairing frequency of homologous transgenic sequences as well
as for an increased colocalization frequency with endogenous
heterochromatin. GFP-lac repressor proteins tagging such loci
may further enhance their tendency for homologous
association. Future studies will show whether DNA
methylation and histone modifications have an impact on
homologous pairing and heterologous association of interstitial
tandem repeats and whether such loci represent hot spots for
somatic recombination, for example after genotoxin exposure.
We thank Jörg Fuchs, Rigomar Rieger, Jerzy Paszkowski and the
anonymous referees for helpful suggestions and critical reading of
the manuscript and Joachim Bruder for technical assistance. This
work was supported in part by grants of the Deutsche
Forschungsgemeinschaft (Schu 951/8-2 and 10-1) to I.S. and by a
grant from the Plant Genome Research Program of NSF (#0077617)
to N.K. and E.L.
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for NOR-bearing chromosomes.
Journal of Cell Science 118 (16)
Journal of Cell Science