Chromosome Research 11: 227^240, 2003.

227
# 2003 Kluwer Academic Publishers. Printed in the Netherlands

Heterochromatin in interphase nuclei of Arabidopsis thaliana

P. Fransz1, W. Soppe2,3 & I. Schubert2

1
Swammerdam Institute for Life Sciences, University of Amsterdam, 1090 GB Amsterdam, The
Netherlands; Tel.: þ 31 20 5255153; Fax: þ 31 20 5257934; E-mail: fransz@science.uva.nl;
2
Department of Cytogenetics, Institute of Plant Genetics and Crop Plant Research (IPK),
D06466 Gatersleben, Germany; 3 Present address: Max-Planck-Institut fˇr Zˇchtungsforschung,
Carl-von-Linne¤-Weg 10, D-50829 K˛ln, Germany

Key words: Arabidopsis, DNA methylation, epigenetic imprint, heterochromatin, histone modi¢cation,
interphase nucleus

Abstract

The eukaryotic nucleus represents a complex arrangement of heterochromatic and euchromatic domains,
each with their speci¢c nuclear functions. Somatic cells of a multicellular organism are genetically identical,
yet they may differ completely in nuclear organization and gene expression patterns. Stable changes in gene
expression without modifying the sequence are the result of epigenetic changes and include covalent modi-
¢cations in cytosine residues of DNA and in histone tails giving rise to altered chromatin protein
complexes, remodeling of chromatin and changes in chromatin compaction. Large-scale differences in
chromatin structure are visible at the microscopic level as euchromatin and heterochromatin. Arabidopsis
thaliana chromosomes display a relatively simple distribution of euchromatic and heterochromatic
segments overlapping with gene-rich and repeat-rich regions, respectively. Recently, we have shown that
Arabidopsis provides a well-de¢ned system to study individual chromosomes and chromatin domains
in interphase nuclei as well as the relationship between chromatin condensation and epigenetic mechanisms
of gene silencing. This overview focuses on the organization and composition of heterochromatin in
Arabidopsis nuclei.

Introduction aceto carmine and later con¢rmed with other

The term heterochromatin is widely used to desig-
nate condensed chromatin regions, inert chro-
matin or chromatin containing silenced genes.
Originally heterochromatin is described as chro-
mosome segments or chromosomes that remain
condensed (positive heteropycnosis) during
interphase and stain di¡erently from the mass of
nuclear euchromatin (Heitz 1928). The dis-
crimination between euchromatin and hetero-
chomatin was initially based on light microscopic
observations using chromatin-speci¢c dyes such as
techniques, such as £uorescence in-situ hybridi-
zation (FISH). Since the ¢rst description, several
additional features have been assigned to hetero-
chromatin (Karpen & Allshire 1997, Hennig 1999,
Heniko¡ 2000): (1) cytologically, as intensely
stained, highly condensed chromosomal regions,
(2) molecularly, as chromosome segments con-
taining abundant tandemly repeated DNA,
(3) biochemically, as a DNA^protein complex
that is relatively insensitive to DNase I, enriched
in heavily methylated DNA and histones H3
methylated at position lysine 9, (4) physically, as
228
chromatin with regularly arranged nucleosomes,
(5) functionally, as largely inactive in transcrip-
tion, late-replicating, rarely involved in meiotic
recombination but frequently involved in error-
prone post-replicative repair. Although the
molecular mechanism is not clear, hetero-
chromatin is involved in centromere function and
position e¡ect variegation. Unless otherwise
stated, we use the term heterochromatin to des-
ignate microscopically discernible, condensed
chromatin structures. We discuss position, for-
mation, structure and function of heterochromatin
in Arabidopsis thaliana in relation to the recently
postulated ‘histone code’ and chromatin-
remodeling processes. Special attention is paid to
the relationship between repetitive sequences,
DNA methylation, histone modi¢cation and
maintenance of heterochromatin.
Chromosomal distribution of heterochromatin
Heterochromatic segments are very distinct in
meiotic mid-prophase cells, when the homologs
are fully paired, forming traceable, linear struc-
tures. In Arabidopsis they are remarkably small,
occupying only 7.4% of the total length of the
pachytene complement (Fransz et al. 1998). In
comparison, 15% of the pachytene chromosomes
of Medicago truncatula (genome size *500 Mb) is
heterochromatic (Kulikova et al. 2001), while, in
tomato (genome size *960 Mb), this percentage is
24% (de Jong et al. 1999). The packaging ratio of
heterochromatin in pachytene chromosomes of
Arabidopsis (genome size 130 Mb) ranges from 0.7
Mb/mm (knob hk4S) to 2.24 Mb/mm (nucleolus
organizing region, NOR), while euchromatin
ranges between 170 and 540 kb/mm with an
average of 330 kb/mm (Fransz et al. 2000). Mitotic
metaphase chromosomes are about 2.5 mm and
contain on average 10.4 Mb per mm.
Heterochromatin is predominantly con¢ned to
the pericentromeric regions of all chromosomes
and the nucleolus organizing regions (NORs) of
chromosomes 2 and 4 (Koornneef et al. 2003 (this
issue)), which comprise the ribosomal 18S, 5.8S
and 25S units, together known as 45 S rDNA.
Pericentromeric regions and NORs contain all
major tandem repeats of the Arabidopsis genome
(Heslop-Harrison, et al. 2003 (this issue)). The
P. Fransz et al.
NORs 2 and 4 each span in total 3.5^4.0 Mb in
ecotype Columbia (Copenhaver & Pikaard 1996).
However, at least in ecotype C24, NOR4 is
signi¢cantly larger than NOR2 (Fransz unpub-
lished data). About 1000 copies of 5S rDNA
genes are located within the centromeric regions
of chromosomes 3, 4 and 5 (Campell et al. 1992,
Cloix et al. 2000). Despite their position in
heterochromatin, the ribosomal genes are
amongst the most highly expressed genes.
Apart from the major heterochromatin seg-
ments, some ecotypes have interstitial hetero-
chromatic segments. For example, Columbia and
Wassilevskija contain a heterochromatic knob,
hk4S, in the short arm of chromosome 4. This
knob has originated from the pericentromere
after a paracentric inversion event (Fransz et al.
2000). Similar to the pericentromeric region the
knob accommodates many dispersed transposable
elements (TEs) and other repetitive elements
(CSHL/WUGSC/PEB Arabidopsis Sequencing
Consortium 2000), including a 1.9-kb tandem
repeat, located in the distal half of the knob.
The arrangement of tandem repeats £anked by
dispersed TEs resembles the organization of
heterochromatic knobs in maize (Ananiev et al.
1998). However, unlike most maize knobs, the
1.9-kb repeat region of hk4S is not interrupted by
other repeats.
Another interstitial heterochromatic segment
contains multiple 5S rDNA sequences and maps
interstitially in the long arm of chromosome 3 of
ecotype Landsberg erecta (Fransz et al. 1998).
This ecotype was derived from Landsberg (LER)
as a mutant carrying the erecta mutation after
irradiation experiments (Re¤dei 1962). Because
LER lacks a 5 S rDNA locus on chromosome 3,
this heterochromatic segment was probably
duplicated from the 5 S rDNA locus of either
chromosome 4 or 5. The extent of this duplication
and whether it is accompanied by any chromo-
somal rearrangement is unknown.
Pericentromeric heterochromatin
The centromere region of the Arabidopsis chro-
mosomes consists of three domains. The cen-
tromere core, which accommodates the functional
centromere, mainly consists of the 180-bp tandem
Heterochromatin in Arabidopsis
repeats spanning about 1.3^2.1 Mbp (Haupt et al.
2001). It is £anked by heterochromatic domains
that contain many transposons and other dis-
persed repeats. Intriguingly, the tripartite cen-
tromeric regions of all chromosomes in ecotype
WS appear to be equal in size, spanning *4.2 Mb,
except for CEN 4, which measures 3.5 Mb (see
Koornneef et al., in this issue). However, if we
consider that the heterochromatic knob hk4S of
this chromosome originates from the peri-
centromere and spans *0.7 Mb, then the size of
CEN4 þ knob hk4S matches the average size of the
Arabidopsis centromere regions. Although both
the centromere core and £anking regions are
heterochromatic, they have distinct molecular,
structural, functional and genetic properties
(Fransz et al. 2000, Haupt et al. 2001). For
example, no genes have been found in the cen-
tromere, whereas the £anking pericentric domains
contain silent and transcriptionally active genes
(Arabidopsis Genome Initiative 2000, Haupt et al.
2001), albeit at low density (<1 per 100 kb)
compared to euchromatin (*1 gene every 5 kb).
The tripartite organization of centromere core
with tandem repeats, £anked by pericentromeric
heterochromatin segments is in line with the
general model for functional centromeres (Choo
2001). In comparison, the centromere region of
Drosophila also displays three domains, where the
central and £anking domains are referred to as
a- and b-heterochromatin, respectively, (Miklos &
Cotsell 1990). In Drosophila, the a-hetero-
chromatin contains several highly repetitive
satellite DNA sequences and is genetically inert,
whereas b-heterochromatin, which is discernible
only in polytene chromosomes, contains middle
repetitive DNA, interspersed with actively tran-
scribed genes (Lohe & Hilliker 1997, Eberl et al.
1993). Unlike Arabidopsis, a remarkable similarity
in gene density has been observed between
b-heterochromatin and the adjacent euchromatin
(Miklos & Cotsell 1990), indicating comparable
transcriptional potency. Another di¡erence from
Arabidopsis is the presence of chromosome-spe-
ci¢c satellite repeats in the centromere region of
Drosophila. With the exception of the ribosomal
gene clusters no tandem arrays of chromosome-
speci¢c repeats have been found in Arabidopsis. It
remains to be proven whether the (peri)cen-
tromeric domains of Arabidopsis are structurally
229
and functionally equivalent to a- and b-hetero-
chromatin in Drosophila.
Heterochromatin in interphase nuclei
In many plant species, interphase heterochromatin
is visible as discrete, intensely stained regions,
known as chromocenters. As early as 1907, the
number of Arabidopsis chromosomes was inferred
from the maximum number of chromocenters,
after staining nuclei with hematoxylin (Laibach
1907). Chromocenters can easily be observed as
dark spots with phase-contrast microscopy or as
bright DAPI-positive domains with £uorescence
microscopy. Even in living cells, chromocenters
can be visualized in transgenic plants containing
histone H2B conjugated with yellow £uorescent
protein (Figure 1). Chromocenters are positioned
at the nuclear periphery and the nucleolus. The
number of chromocenters per nucleus varies
between 4 and 10 with an average of 8. FISH
analyses of chromosome domains (Fransz et al.
2002) have demonstrated that individual inter-
phase chromosomes are organized as a hetero-
chromatic chromocenter with emanating
euchromatic loops (Figure 2A, B). The chromo-
centers contain all major tandem repeats and the
majority of the dispersed pericentric repeats,
including transposons and non-transposon
repeats. Euchromatic loops, in contrast, are gene-
rich. They range in size from 200 kb up to an entire
chromosome arm. The territory of an Arabidopsis
chromosome, which comprises on average 25 Mb
of DNA with 5200 genes (Arabidopsis Genome
Initiative 2000), is formed by its chromocenter and
the corresponding euchromatic loops. In com-
parison, the average human chromosome terri-
tory, is ¢ve times larger (130 Mb) but contains only
*1700 genes. This di¡erence underlines the small
numbers of repetitive sequences within the Ara-
bidopsis genome and, consequently, the low
content of heterochromatin in the Arabidopsis
nucleus. The organization of euchromatin loops
around chromocenters implies that some gene-rich
regions that are euchromatic in the pachytene
chromosome may also colocalize with chromo-
centers and form attachment sites of the loops to
the heterochromatin. This was indeed observed
when BACs from di¡erent euchromatic regions
230
Figure 1. Confocal images of an interphase nucleus in a mesophyll cell isolated from a transgenic plant containing a histone H2B::YFP
construct. Fluorescent chromatin is strong in chromocenters.
were used as probes for FISH. Colocalization of
DNA sequences with the chromocenter of chro-
mosome 4 varies between di¡erent chromosomal
regions and between cells (Table 1). Whether the
position of gene regions close to chromocenters is
accompanied by gene repression remains to be
elucidated.
The nuclear phenotype
Most somatic cells of a multicellular organism
contain the same DNA sequences but may di¡er
considerably in nuclear organization due to the
dynamic properties of chromatin. The nuclear
phenotype is a cytological characterization of the
interphase nucleus and comprises a number of
parameters. It provides valuable information
about the relationships between nuclear organi-
zation, gene regulation and cell identity. The
cytological parameters include global features,
such as proportion and distribution of hetero-
chromatin, DNA methylation and histone
modi¢cation (see below), but may also relate to
small-scale aspects, such as local chromatin
condensation or gene position. For example, it is
demonstrated that, in human cells, the frequency
with which a chromosome region is present as a
decondensed chromatin loop is cell-type depen-
dent and appears to be related to the number of
active genes in that region (Volpi et al. 2000).
P. Fransz et al.
A quantitative parameter of nuclear phenotype is
the heterochromatin fraction which is measured
from the area and staining intensity of the chro-
mocenters in relation to that of the entire nucleus
(Soppe et al. 2002). Although the method
generates relative values it allows comparing
heterochromatin fractions between di¡erent cell
types and between mutants within a species. We
analysed nuclei from Columbia and a hypo-
methylation mutant, ddm1, which display a lower
content of heterochromatin (see below). The
relative heterochromatin fraction in the hypo-
methylation mutant is signi¢cantly (*30%) lower
than in the wild type (Figure 3). Thus, the method
allows the quanti¢cation of large-scale changes in
chromatin structure, which, in this case, are due to
a decrease in DNA methylation.
The nuclear phenotype in gene-silencing mutants
DNA methylation at cytosine residues is essential
for epigenetic regulation of developmental pro-
cesses in most higher eukaryotes. Approximately
80% of cytosines in CpG dinucleotides in plant
genomes is methylated (Finnegan et al. 1998).
DNA methylation is involved in transcriptional
gene repression and supposed to be targeted
predominantly to heterochromatic regions
(Martienssen & Colot 2001). In Arabidopsis, the
majority of methylated cytosines is found within
Heterochromatin in Arabidopsis 231

Figure 2. Chromocenter-loop model. DAPI-stained image and diagram of a wild-type interphase nucleus (A) with nucleolus (N) and
nine chromocenters (CC). A chromosome territory is magni¢ed (B) showing a repeat-rich chromocenter (gray circle) with emanating
euchromatic loops (gray lines). Note that pericentromeric regions, containing transposons (white ovals), and non-transposon regions
(thick black lines) colocalize with the chromocenter. (C) Diagram of a chromosome territory in a hypomethylation mutant containing
a small chromocenter. Pericentromeric transposons are still in the chromocenter whereas pericentromeric non-transposons are
euchromatic.
232 P. Fransz et al.

Table 1. Percentage of colocalization of gene-rich regions from and have only about 30% of the wild-type DNA
the short arm of chromosome 4. methylation. They are among the strongest gene-
silencing mutants. Both mutants show the same
Regiona Position on 4L Colocalization with CC4 (n)
deviating nuclear phenotype with fewer and size-
rDNA Distal 98% (230) reduced chromocenters (Figure 4C, D). The
F6N15 Subdistal 85% (80) decrease in heterochromatin is accompanied by a
8B1b (WS) Mid 50% (20)
drastic decrease of methylated DNA at the
T4B21 (WS)c Proximal 44% (72)
T4B21 (LER) Proximal 25% (55) remaining chromocenters. The phenotype is stably
inherited in subsequent generations and in dif-
a
The applied probes are BACs (F6N15 and T4B21) and a YAC ferent ecotype backgrounds. Nuclei of the double
(CIC8B1). The rDNA region is detected with the 45S rDNA mutant ddm1-2/met1 show an even stronger
sequence. b8B1 covers the heterochromatic knob hk4S, which
is present in WS, but absent in LER. cIn WS, the T4B21 region
reduction in chromocenter size as well as in global
lies in between the knob and the pericentromeric heterochromatin. DNA methylation. The decrease in number and
size of chromocenters is con¢rmed by the quan-
titative analysis of the relative heterochromatin
centromere-associated tandem repeats, rDNA fractions of ddm1-2, met1 and the double mutant
arrays, and transposons or retrotransposon- (Soppe et al. 2002). The latter shows about 50%
derived elements. Immunolabeling of interphase reduction in heterochromatin.
nuclei with antibodies against 5-methyl cytosine The mutant ddm1-5 is allelic to ddm1-2, but
demonstrates that chromocenters contain most of considered to represent a null allele, since DDM1
the heavily methylated DNA (Figure 4A, B), transcripts are not detectable (Mittelsten Scheid
con¢rming the molecular data. Chromocenters, et al. 2002). The e¡ect of the ddm1-5 mutation on
therefore, likely represent transcriptionally silent the nuclear phenotype is even more severe than
domains. The ‘Decrease in DNA Methylation’ that of ddm1-2. Chromocenters are highly disin-
genes DDM1 and DDM2 (or MET1) play an tegrated and the 180-bp repeats may dislocate
important role in DNA methylation of plants from chromocenters, suggesting a decondensation
(Vongs et al. 1993, Ronemus et al. 1996). DDM1 is of the centromeric core (Mittelsten Scheid et al.
a SWI/SNF-like chromatin remodeling protein 2002). These data indicate a strong correlation
(Jeddeloh et al. 1999), while MET1 is a main- between the level of DNA methylation and the
tenance methyltransferase. The hypomethylation organization of heterochromatin. Incorporation
mutants, ddm1-2 and met1, are point mutations of 5-azacytidine into DNA of mammalian cells

Figure 3. Histogram showing the relative heterochromatic content in wild-type (gray) and ddm1 mutant (black) plotted against the
number of nuclei.
Heterochromatin in Arabidopsis
also revealed a negative e¡ect of DNA hypo-
methylation on heterochromatin condensation
(Haaf & Schmidt 1989), but the direct cause of the
decondensation is still unclear. The hypomethy-
lation mutants of Arabidopsis demonstrate that
DNA methylation a¡ects the formation of het-
erochromatic structures. However, in some cases,
heterochromatin is not a¡ected by DNA methy-
lation. For example, a small fraction of nuclei in
the hypomethylation mutants displays the wild-
type phenotype with conspicuous chromocenters
(Mittelsten Scheid et al. 2002, P. Fransz unpub-
lished data). These cells have reduced DNA
methylation but are capable of maintaining wild-
type levels of microscopically visible hetero-
chromatin, suggesting that, in some cases, the role
of DNA methylation in heterochromatin forma-
tion may be compensated by some other epigenetic
mechanism. This situation is reminiscent of
Drosophila nuclei, which contain visible hetero-
chromatin but lack persistent DNA methylation.
The MOM (Morpheus’ molecule) gene product
is essential for transcriptional gene silencing but
does not a¡ect the DNA methylation level
(Amedeo et al. 2000). Several heavily methylated
genes, including silent transgenes and endogenous
inactive TSI (transcriptionally silent information)
elements, became active after mutation of the
MOM gene or after depletion of its transcript
(Amedeo et al. 2000, Steimer et al. 2000). In
contrast to the situation in ddm1 and met1,
heterochromatin in nuclei of mom1 reveals a wild-
type appearance. It is particularly remarkable that
several re-activated genes in mom1 are still posi-
tioned within heterochromatic structures (Probst
et al. 2002). This suggests that: (1) hetero-
chromatin structure, in some cases, is not su⁄cient
to repress transcription of repetitive genes, and (2)
MOM1 may be essential to silence certain hetero-
chromatic genes. It underlines the fact that genes
in heterochromatin are not inactive per se and it
should be kept in mind that some Drosophila genes
are only active in a heterochromatic environment
(Eberl et al. 1993). A surprising feature in the
double mutant mom1/ddm1-5 is that, in some
nuclei, all chromocenters are clumped together
forming an aggregation of heterochromatin
(Mittelsten Scheid et al. 2002). Apparently,
absence of MOM1 alone does not a¡ect the
nuclear organization of heterochromatin but, in a
233
ddm1-5 mutant background, MOM1 is involved in
heterochromatin structure. MOM1 and DDM1
may therefore have complementary function in the
nuclear organization of chromatin.
Other less-extensively studied mutants of gene
silencing reveal a nuclear phenotype comparable
to either wild type or ddm1. The kyp2 mutant is
de¢cient for a methyltransferase speci¢c to histone
H3 at lysine 9 (H3K9) (Jackson et al. 2002). Nuclei
of kyp2 have a wild-type appearance of hetero-
chromatin, showing strong DNA methylation at
chromocenters (Jasencakova et al. 2002). Simi-
larly, cmt3, which lacks a functional chromo-
methyltransferase (Heniko¡ & Comai 1998),
shows wild-type heterochromatin levels (P. Fransz
unpublished data). In contrast, heterochromatin in
the silencing mutants hog1 and sil1 (Furner et al.
1998) is decreased (P. Fransz unpublished data).
Both hog1 and sil1 are monogenic recessive
mutants but the gene products are unknown. The
hog1 mutant reduces DNA methylation at ribo-
somal DNA and a transgene locus C containing
multiple copies of the genes CHS, NPT and HPT
(Furner et al. 1998). Although hog1 resembles
ddm1 in DNA methylation level and gene silen-
cing, hog1 is not a ddm1 allele. The sil1 mutant, on
the other hand, does not show reduction of DNA
methylation and releases gene silencing of only the
NPT and HPT genes.
Are transposable elements anchoring points of
euchromatin loops to heterochromatic
chromocenters?
The reduction of heterochromatin in nuclei of the
hypomethylation mutants, ddm1-2 and met1,
implies that certain DNA regions are no longer in
heterochromatin after decrease of DNA methy-
lation. Whereas major tandem repeats remain
largely within the chromocenters of the hypo-
methylated nuclei (Figure 4E, F), pericentromeric
regions with dispersed repeats were found pre-
dominantly outside chromocenters. Further ana-
lysis by FISH on hypomethylated nuclei revealed
that high-copy transposon elements from di¡erent
families, including Ty3-gypsy, Ty1-copia and
miniature inverted-repeat transposable element
(MITE), still co-localize within chromocenters
(Figure 4G, H). Non-transposon elements,
234 P. Fransz et al.
Heterochromatin in Arabidopsis 235

however, appeared dislocated into euchromatin
(Soppe et al. 2002). The results indicate that the
organization of pericentromeric chromatin
depends on the type of repeats they contain. We
propose that, in hypomethylated nuclei, (1) tan-
dem repeats form heterochromatic chromocenters,
(2) non-transposon sequences are in euchromatin
loops, and (3) high-copy transposon elements are
located in heterochromatin at the bases of these
loops (Figure 1C). It is tempting to speculate that
transposon elements are also anchor points for
gene-rich euchromatin loops to heterochromatin.
In this context, it is intriguing to ¢nd that the
dispersed multicopy transposons, Emi12 and
AthE1.4, which colocalize at chromocenters in
both wild-type and hypomethylation mutants
(Soppe et al. 2002), are frequently mapped to gene-
rich regions. As in the wild type, we observed a
chromocenter-loop organization of chromosome
arm 4S in ddm1 nuclei, suggesting that, in hypo-
methylated nuclei, some regions of the gene-rich
euchromatin remain attached to chromocenters.
Whether these sequences involve transposon ele-
ments is still not known. The inactive single copy
transposon, CAC1, which is present on chromo-
some arm 2L, frequently (83%, n ¼ 78) colocalizes
with the chromocenter in wild-type nuclei (Soppe
et al. 2002). However, in ddm1 nuclei, the trans-
poson is derepressed (Miura et al. 2001), and 68%
(n ¼ 95) of the signals appear within euchromatin,
indicating a dislocation of the reactivated trans-
poson from the chromocenter into euchromatin.
Therefore, if transposon elements are involved in
anchoring euchromatin loops to heterochromatin,
they are presumably not active.
Although the methylation level of the low-copy
non-transposon repeats in ddm1 and met1 is
unknown, their FISH signals follow the same
pattern as the DNA methylation signals (Figure 4I^N).
This suggests that pericentromeric non-transposon
repeats, although being euchromatic, still show
some DNA methylation in ddm1-2 and met1.
Apparently, DNA methylation in this case a¡ects
heterochromatin structure but is not su⁄cient to
form heterochromatin. This is not surprising
because euchromatin of wild-type nuclei may also
contain methylated DNA. It is therefore con-
ceivable that a minimal level of DNA methylation
in pericentromeric non-transposon elements is
required for heterochromatin formation.
Maintenance of heterochromatin and epigenetic
imprinting
The bulk of DNA methylation activity occurs via
maintenance of DNA methylation, which is
established directly after DNA replication at
hemimethylated sites, when existing methylation
marks are copied to the new DNA strands. It
involves DDM1, MET1 and CMT3, a methyl-
transferase containing a chromodomain (Heniko¡
& Comai 1998). MET1 methylates CpG sites, the
majority of methylated sites, while CMT3, which
is unique to plants, maintains methylation of
CpNpG and asymmetric sites (Lindroth et al.
2001). Mutants lacking one of these genes show
reduced DNA methylation levels. Full recovery of
DDM1 and MET1 activity in the respective
mutants is established after crossing with wild-
type plants. However, the DNA methylation level
in the F1 generation appeared to be intermediate
between the mutant and the wild-type parents
(Vongs et al. 1993, Finnegan et al. 1998). Immediate
remethylation does not take place because the
mutant-derived chromosomes lack a methylation
imprint that is necessary for maintenance of
DNA methylation. Remethylation requires de-
novo DNA methylation activity established by
‘Domains Rearranged Methylase’ (DRM) (Cao &

3
Figure 4. FISH images and immunolabeling patterns presenting distinct nuclear phenotypes of wild-type and mutant nuclei. (A,B)
Wild-type and (C,D) ddm1 nuclei labeled with anti-5-methylcytosine (green) and stained with DAPI (blue). (E,F) Wild-type and
(G,H) ddm1 nuclei probed with transposon Ta1 (green) and pericentromeric BAC F28D6 repeats (red). (I,J) Wild-type and (K^N)
ddm1 nuclei probed with pericentromeric BAC F17A20 repeats (green) and immunolabeled with anti-5-methylcytosine (green).
Magni¢cation (M,N) of the ddm1 nucleus shows partly overlapping patterns of repeats and DNA methylation outside chromocenters
(blue circles). (O,P) F1 nucleus heterozygous for DDM1 probed with 45 S rDNA (red) and immunolabeled with anti-5-methylcytosine
(green). Arrow indicates the mutant-derived chromocenters. (Q,R) Wild-type nucleus probed with the 180-bp centromere repeat (red)
and immunolabeled with antiserum against acetylated histone H4Ac5 (green). (S) Wild-type nucleus immunolabeled with antiserum
against dimethylated histone H3K9 (red). (T) Wild-type nucleus probed with the 180-bp centromere repeat (red) and immunolabeled
with antiserum against methylated histone H3K4. (For materials and methods, see Soppe et al. 2002.)
236
Jacobsen 2002). Because repeated backcrossing to
a wild-type parent only slowly increased the level
of methylation at tandem repeats in each gen-
eration, it was concluded that de-novo methylation
activity at these sites is rather slow or non-existent
(Kakutani et al. 1999).
The di¡erent degree of methylation of the
parental genomes in F1 nuclei of crosses between
wild-type and hypomethylation mutant is re£ected
in the nuclear phenotype. About half of the
chromocenters have a wild-type appearance, while
the other half are smaller. The wild-type-derived
chromosomes show heavily methylated chromo-
centers, whereas mutant-derived chromocenters
lack clear DNA methylation signals (Figure 4O,
P). Quanti¢cation of heterochromatin content
revealed an intermediate level of heterochromatin
in the F1 generation compared to the mutant and
wild-type parents (Soppe et al. 2002). The wild-
type- and mutant-derived chromocenters di¡er in
the presence of pericentric non-transposon
sequences. Apparently, DDM1 and MET1 are not
su⁄cient to restore heterochromatin in pericentric
regions containing non-transposon repeats once
DNA methylation is lost. We conclude that het-
erochromatin in these regions acts as a self-
perpetuating chromatin state and requires a DNA
methylation imprint.
Heterochromatin and histone code
Gene expression is the result of a complex
molecular interplay between DNA sequence and
chromatin-associated proteins. Covalent modi¢-
cations in histone tails give rise to altered
chromatin protein complexes, remodelling of
chromatin, changes in chromatin density and,
¢nally, altered accessibility of the DNA to the
transcription machinery. At the microscope level,
the large-scale di¡erences are manifested as
heterochromatin and euchromatin. Arabidopsis
nuclei display distinct genome-wide labeling
patterns of modi¢ed histones that correspond to
heterochromatin and euchromatin. Chromo-
centers contain high levels of methylated histone
H3 at lysine position 9 (H3K9) and low levels of
acetylated histone H4 at position K5 and K8. In
contrast, euchromatin regions show hyper-
acetylated histones H4 at position K5, K12, K8
P. Fransz et al.
and K16, but also hypermethylated histone H3K4
(Figure 4Q^T, Fransz et al. 2002, Soppe et al.
2002, Jasencakova et al. 2002, Probst et al. 2002).
The distinct distribution of these modi¢ed his-
tones within heterochromatin and euchromatin
underscores the sharp boundaries of hetero-
chromatic chromocenters in Arabidopsis nuclei
and hence the distinct separation of functional
subdomains within a chromosome territory.
Genome-wide immunolabeling of acetylated
histones in mitotically active cells has further
demonstrated that the chromocenters contain low
levels of histones H4 acetylated at position K12
and K16 and of histone H3 acetylated at K9 and
K18. Only during and shortly after DNA repli-
cation, are H4K16 and H3K9 strongly acetylated
within chromocenter regions. However, after
postreplicative repair, maintenance of DNA
methylation at CpG sites and methylation of
H3K9, these lysine residues become deacetylated
to a level below that of euchromatin (Jasencakova
et al. 2002).
In DNA hypomethylation mutants, histone
methylation at H3K9 is low and barely clustered at
the remnant heterochromatic chromocenters
(Soppe et al. 2002). The decrease of hetero-
chromatin in the hypomethylation mutants is
mainly caused by dislocation of pericentric non-
transposon regions away from heterochromatin.
Apparently, these regions have lost methylated
H3K9 and gained methylated H3K4. In nuclei of
F1 plants heterozygous for ddm1 (or met1), one
half of the chromocenters derived from the wild
type shows strong DNA and H3K9 methylation,
whereas chromocenters from the hypomethylated
parent do not. Thus, DDM1 and MET1 of the
heterozygous F1 plants apparently cannot recover
histone methylation in the pericentric non-trans-
poson repeats from mutant-derived chromosomes.
Hypermethylation of H3K9 is restricted to
chromocenters with strongly methylated DNA
derived from the wild-type parent, indicating that
a DNA methylation imprint is responsible for
strong methylation of H3K9 in heterochromatin.
What determines heterochromatin in Arabidopsis?
Microscopically visible heterochromatin displays
di¡erent features in Arabidopsis nuclei. These
Heterochromatin in Arabidopsis 237

seem to depend on the underlying DNA sequences
but also on the presence of proteins involved in
modi¢cation of DNA and chromatin. The ques-
tion arises, what is required to form hetero-
chromatin in Arabidopsis. Since we have shown
that the heterochromatin state is epigenetically
transmitted, the question may be rephrased into
‘What is required to maintain heterochromatin?’.
Wild-type nuclei contain well-de¢ned hetero-
chromatin regions, rich in repetitive sequences,
including tandemly arranged repeats, transposon
elements and non-transposon repeats. In addition,
some sequences from gene-rich regions may also
associate with chromocenters as anchor points of
the euchromatic loops. In mutants defective for
speci¢c chromatin proteins, however, the corre-
lation between repeats and heterochromatin
becomes less clear. Based on the presented data we
classi¢ed Arabidopsis DNA sequences into four
groups according to their position relative to
heterochromatin in wild type and in chromatin
mutants (Table 2). Tandemly arranged repeats
colocalize with heterochromatin, irrespective the
methylation levels of DNA or histone H3 at lysine
K9. These levels vary depending on the mutant
background. At least one exception to this rule
forms the ddm1-5 mutant, which shows decon-
densed tandem repeat regions. Regions with
multicopy transposon elements follow the same
pattern as tandem repeats and colocalize with
chromocenters irrespective of DNA or H3K9
methylation. Pericentric non-transposable ele-
ments, on the other hand, are in heterochromatin
or in euchromatin, depending on the DNA

Table 2. Cytological and biochemical properties of genomic regions in interphase nuclei of wild-type and epigenetic mutants.

Backgrounda Region Heterochromatin 5mC H3metK4 H3metK9 H4Ac5/Ac8

WT Gene-rich  Lowb High Low High

WT Pericentric non-TE þ High Low High Low
WT Pericentric TE þ High Low High Low
WT Tandem repeat þ High Low High Low
ddm1-2 Gene-rich  Low High Low High
ddm1-2 Pericentric non-TE  Low High Low High
ddm1-2 Pericentric TE þ Low Low Low Low
ddm1-2 Tandem repeat þ Low Low Low Low
met1 Gene-rich  Low High Low High
met1 Pericentric non-TE  Low High Low High
met1 Pericentric TE þ Low Low Low Low
met1 Tandem repeat þ Low Low Low Low

kyp2 Gene-rich  Low High Low High

kyp2 Pericentric non-TE þ High Low Low Low
kyp2 Pericentric TE þ High Low Low Low
kyp2 Tandem repeat þ High Low Low Low
ddm1-5 (type 4)c Gene-rich  Low High Low High
ddm1-5 (type 4) Pericentric non-TE nd nd nd nd nd
ddm1-5 (type 4) Pericentric TE nd nd nd nd nd
ddm1-5 (type 4) Tandem repeat (180 bp) /þd Low High/Low Low Low
ddm1-5 (type 4) Tandem repeat (45S rDNA) þ Low Low Low Low

mom1e Gene-rich  Low High Low High

mom1 Pericentric non-TE þ High Low High Low
mom1 Pericentric TE þ High Low High Low
mom1 Tandem repeat þ High Low High Low
a
Data are from Mittelsten Scheid et al. 2002 (WT, ddm1-5), Fransz et al. 2002 (WT), Soppe et al. 2002 (WT, ddm1-2, met1), Probst et al.
2002 (WT, ddm1-5, mom1) and Jasencakova et al. 2002 (WT, kyp2). bLow levels of labeling in one region may differ from low levels
in other regions. cType 4 nuclei display the most severe nuclear phenotype in the mutant ddm1-5 (Mittelsten Schied et al. 2002). The
detection of non-TEs and TEs was not established in this mutant. However, pericentromeric non-TEs are likely in euchromatin. dThe
180-bp repeat shows dispersed signals in euchromatin as well as condensed signals at chromocenters. e The non-overlapping patterns
of repeats and acetylated histones was inferred from the nuclear phenotype, which resembles the wild type (Probst et al. 2002).
238
methylation status but not on histone H3K9
methylation (see kyp2 mutant). Gene-rich regions
always have low levels of DNA methylation and
histone H3K9 methylation. Although these
regions are predominantly in euchromatin, some
sequences may colocalize to heterochromatin in
wild type as well as in hypomethylation mutants.
We propose that speci¢c sequences, most likely
transposable elements, may be responsible for
anchoring euchromatin loops to heterochromatin.
It has been suggested that DNA methylation plays
a role in the defense mechanism against parasitic
sequence elements, such as transposons, in par-
ticular by identifying them and maintaining their
heterochromatic state (Yoder et al. 1997). In this
view it is tempting to speculate that the mechanism
of inactivating transposable elements may have
evolved together with a mechanism for gene
silencing by recruiting adjacent genes to hetero-
chromatic domains.
From the above, we may conclude that,
depending on the epigenetic conditions, all types of
DNA sequences may become part of hetero-
chromatin or euchromatin (Tables 1 & 2). The
only correlation that remains in all mutants is the
negative correlation between heterochromatin and
methylated histone H3K4 and acetylated histones
H4Ac5 and H4Ac8. These modi¢ed histone levels
are always low in heterochromatin and high in
euchromatin, irrespective of the epigenetic mutant
background and irrespective of the DNA
sequence. In human cells, methylation of H3K4,
which is established by the methyltransferase
SET9, inhibits methylation at H3K9 and precludes
association of histone deacetylase (HDAC)
complexes with the H3 tail and facilitates trans-
cription (Maison et al. 2002). It is likely that a
similar mechanism is present in Arabidopsis to
prevent formation of heterochromatin. The for-
mation of microscopically visible heterochromatin
may be dependent on the ratio between methylated
H3K9 and methylated H3K4. Once hetero-
chromatin is formed, it is maintained via an
epigenetic imprint. Methylated cytosine residues
may provide the epigenetic imprint. In addition,
histone methylation has been shown to be a stable
epigenetic imprint. Alternatively, protein com-
plexes containing polycomb group (PcG) proteins,
P. Fransz et al.
may also form a stable chromatin imprint.
Moreover, some PcG proteins have been
demonstrated to form complexes with histone H3
methyltransferase (Czermin et al. 2002, Sewalt et al.
2002). Finally, association of sequence-speci¢c
RNA molecules may be involved in formation and
maintenance of heterochromatin (Maison et al.
2002, Volpe et al. 2002).
Originally, heterochromatin was subdivided
into two classes, constitutive and facultative
heterochromatin, depending on its persistent
presence throughout the cell cycle (Brown 1966).
Constitutive heterochromatin includes pericen-
tromeric and NOR-associated heterochromatin
because these regions remain heterochromatic.
Facultative heterochromatin varies in its state in
di¡erent cell types or from one homologous
chromosome to another and is heteropycnotic in
special cell types. Francastel et al. (2000) described
facultative heterochromatin as that fraction of
chromatin that is compact and transcriptionally
inactive in one cell lineage, but may be decon-
densed and active in another. We propose that, in
Arabidopsis, the term constitutive hetero-
chromatin be applied to regions with tandem
repeats or high-copy transposons. Their hetero-
chromatic state is not controlled by DNA
methylation. Facultative heterochromatin, on the
other hand, may comprise non-transposons, low-
copy transposon or small gene-rich regions. Their
heterochromatic state is under the epigenetic
control of DNA methylation.
Acknowledgements
We gratefully acknowledge R. van Driel,
O. Mittelsten-Scheid and A. Probst for critically
reading of the manuscript, R. Martienssen,
J. Paszkowski, S. Jacobsen and C. Spillane for
providing gene-silencing mutants, F. Berger for
providing the H2B::YFP transformant, Monica
Ru⁄ni-Castiglione for providing anti-5-methyl-
cytosine and B. M. Turner for providing antisera
against acetylated histones. We acknowledge
¢nancial support from the Deutsche Forschungs
Gemeinschaft (SCHU 951/9-2, I.S. and FR 1497/
1-1, P.F.).
Heterochromatin in Arabidopsis
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