Update on Heterochromatin
Heterochromatin in Animals and Plants.
Similarities and Differences
Zoya V. Avramova*
School of Biological Sciences, Manter Hall, University of Nebraska, Lincoln, Nebraska 68588
Gene function is subjected to the effects of surrounding chromatin. The nature of these effects may
be epigenetic occurring in some cells, but not others,
of the same genetic background. Epigenetic regulatory mechanisms remain an enigma, but recent studies have provided informative insights into the molecular basis underlying them (for review, see
Henikoff et al., 2001; Martienssen and Colot, 2001;
Reik et al., 2001).
Inside the nucleus, the three levels of structural
compaction of DNA are seen as 11-, 30-, and 300-nm
fibers, the latter representing the folding of the 30-nm
chromatin fiber into loops attached to the nuclear
matrix. In addition, regions of dense heterochromatin
masses scattered throughout the interphase nucleus
have been known for over 100 years. Only recently
has some understanding of the mechanisms of its
formation and propagation been achieved. Genes
coding for proteins found in heterochromatin provide our major source of current information on the
structure and function of heterochromatin.
At the cytological level, heterochromatin is seen at
the telomeres, at the centromeric and pericentromeric
regions, at chromosome 4 of Drosophila melanogaster,
along the arms of some mammalian autosomes,
along the arms of the animal Y-chromosome (for
review, see Eissenberg and Elgin, 2000), and the
whole inactive mammalian X chromosome (Park and
Kuroda, 2001). No morphological structures corresponding to heterochromatin can be seen in Saccharomyces cerevisiae. In plants, in addition to the centromeric and pericentromeric regions, heterochromatin
is located at the nucleolar organizer, at the knobs,
and along the maize (Zea mays) B chromosomes (Alfenito and Birchler, 1993; Copenhaver et al., 1999;
Fransz et al., 2000; McCombie et al., 2000).
Heterochromatin is divided into constitutive heterochromatin, containing satellite DNA found usually at the centromeres, and facultative heterochromatin, inactive in a certain cell lineage but expressed
in other lineages. An example for facultative heterochromatin is the mammalian X chromosome, where it
is essential for the inactivation of one of the X chromosomes (Park and Kuroda, 2001).
* E-mail [email protected]; fax 402– 472–2083.
www.plantphysiol.org/cgi/doi/10.1104/pp.010981.
40
Most fascinating is the involvement of heterochromatin in epigenetic silencing phenomena including
repression along extended regions of chromosomes
(around the centromeres and the pericentromeres)
and the inactivation of whole chromosomes (inactive
mammalian X chromosome; for review, see Reik et
al., 2001, and refs. therein). The potential of heterochromatin to silence nearby genes, a phenomenon
known as position effect variegation (PEV), has been
both puzzling and attractive for scientists since its
discovery (for review, see Eissenberg and Elgin,
2000).
Before discussing the specific features of heterochromatin, I would like to draw the attention to a
potential caveat and to an often-encountered misconception. Heterochromatin is not synonymous with
gene silencing, with methylated DNA, or with
deacetylated histones. Although the heterochromatin
of different species may display some, or all, of these
features, the mechanisms responsible for heterochromatin formation, propagation, and silencing may not
be the same as the mechanisms involved in “normal”
silencing of euchromatin genes. Thus, heterochromatin silencing involves large-scale modifications of
chromatin structure, acting as a global silencing
mechanism. “Normal” silencing mechanisms target
specific genes and the scope of chromatin modifications, although not precisely defined, probably does
not expand beyond the promoter and the vicinity of
the silenced gene. Heterochromatin is one among
several epigenetic silencing factors. Cytosine methylation and histone deacetylation are two other such
factors. Although these activities can modify the nucleosomes and alter chromatin structure, none of the
known cytosine methyltransferases or deacetylases
(except Clr3, see further) have been implicated in
either formation or function of heterochromatin. Despite the fact that the heterochromatin of many species contains densely methylated DNA, it is not
known whether methylated DNA can provoke assembly of heterochromatin. The histones in heterochromatin are usually deacetylated, but it is not
known whether the same amino acids are deacetylated as those deacetylated for the purpose of euchromatin gene silencing, neither it is known whether the
same deacetylases function in both types of histone
modification. There is an astonishingly large amount
of histone acetylases and deacetylases in the eukary-
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Heterochromatin in Animals and Plants
otic genomes and it remains to be seen what the
functions of most of them are and whether they
target the same histone amino acids in normal gene
silencing processes and in heterochromatin formation. Therefore, it is not correct to automatically link
eukaryotic gene silencing and associated alterations
in chromatin structure with heterochromatin as it
will be discussed in the chapter of the Polycomb
group (Pc-G) silencers. Recent breakthrough studies
provided first insights into the biochemical activity
of some protein components of heterochromatin and
a molecular basis for its initiation, propagation and
maintenance. These will be discussed in the context
of their validity for plant heterochromatin.
MOLECULAR COMPONENTS OF
HETEROCHROMATIN
The DNA Component
Most of our current knowledge on the nature of the
DNA moiety of heterochromatin comes from studies
of the centromeric, pericentromeric, and knob regions of chromosomes. The densely packed regions
in the nucleus, at the cytological level, are composed
of repetitive DNA at the molecular level. According
to conventional knowledge, highly repetitive DNAs
underlie heterochromatin formation. The real picture
is more complex with a mosaic arrangement of different types of middle repetitive, satellite, and even
unique sequences packaged into heterochromatin
(Copenhaver et al., 1999; Fransz et al., 2000; McCombie et al., 2000). Many of these repeats represent
different types of transposable elements. This issue
will not be discussed in detail because it was recently
reviewed (Henikoff et al., 2001; Martienssen and Colot, 2001). It will be noted, however, that the recently
sequenced centromeric and pericentromeric regions
from Arabidopsis chromosomes (Copenhaver et al.,
1999; Fransz et al., 2000; McCombie et al., 2000) provided first insights into the nature, composition, and
function of these regions at the molecular level. These
results are important not only because they filled a
gap in the current knowledge regarding these structures in plants, but also because they provided answers to longstanding questions of general importance. They demonstrated that the centromere and
the pericentromere are composed of different types
of repeats, are organized differently, have different
condensation properties at the different phases of the
mitosis, and contain different sets of low-copy DNA
sequences (Fransz et al., 2000; McCombie et al., 2000).
Thus, despite appearing simply heterochromatic, the
centromere and the pericentromere are molecularly,
structurally, and functionally different subregions of
the chromosomes.
The repetitive DNA in heterochromatin is usually
methylated, in accordance with a predicted repressive function. Recent reviews on the role of DNA
methylation in mammalian, plant, and fungal epigePlant Physiol. Vol. 129, 2002
netic inheritance are recommended (Martienssen and
Colot, 2001; Reik et al., 2001). It is important to note
that DNA methylation is not conceived as a factor
provoking heterochromatin formation (some species
may lack methylation altogether) but rather as a factor stabilizing heterochromatin structures (for review, see Wolffe and Matzke, 1999).
In summary: (a) in most species, the DNA moiety
of heterochromatin is made of methylated repetitive
DNAs of different types (including mobile elements)
intermixed with low-copy and unique sequences; (b)
a prerequisite for heterochromatin formation appears
to be the structural organization of the repeats rather
than the nature of the particular sequences, or their
repetitive character; and (c) based on the types and
the arrangement of the repetitive DNAs, heterochromatin in plants is similar to the heterochromatin in
animals.
However, at least three features make plant heterochromatin different from the animal heterochromatin: (a) absence of proteins similar to known heterochromatin proteins (see “Note Added in Proof”); (b)
location of potentially active genes in the knob structures and in the pericentromeric regions of plant
genomes; and (c) different chromosomal environments for colinear genes in related species. These
differences raise the following questions: (a) whether
plant heterochromatin DNA functions in a complex
with unknown yet plant-specific proteins (see “Note
Added in Proof”); (b) whether plant heterochromatin
can silence nearby genes; and (c) whether plants have
evolved mechanisms to recruit heterochromatin for
large-scale silencing as animals have. These will be
discussed below.
Protein Components of Heterochromatin
Protein components were discovered about 20
years ago and have attracted attention because of
their role in PEV, a paradigm for the silencing activity of heterochromatin (for review, see Eissenberg
and Elgin, 2000). It is important to note that position
effects result from translocation events, placing a
normally euchromatic gene into a heterochromatin
environment, or from ectopic expression of transgenically introduced genes. Therefore, PEV is not a
“normal” mechanism for silencing euchromatin
genes. This has to be kept in mind when gene silencing is analyzed. Nevertheless, studies of PEV have
provided revealing insights into heterochromatin
properties. The severity of PEV, following specific
gene mutations, has allowed identification of genes
affecting the formation of heterochromatin.
Genetically, about 60 different loci in D. melanogaster have been defined as modifiers of PEV, suppressing [Su(var)] or enhancing [E(var)] variegation.
Only a small fraction has been identified biochemically. Major heterochromatin components are products of two Su(var) genes in D. melanogaster [Su(var)-
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41
Avramova
and Su(var)3–9], their homologs in animals, and in
fission yeast (Schizosaccharomyces pombe; for review,
see Eissenberg and Elgin, 2000). Notably absent from
the databases are homologs of these genes in the
budding yeast, S. cerevisiae and in any plant species.
The biochemical activities of Su(var)2-5 and
Su(var)3-9 were established recently, suggesting a
possible molecular basis for their roles in heterochromatin. The two proteins belong to the superfamily of
the chromodomain proteins (see below). Some of
them belong to plant-specific families, whereas others belong to a family of proteins conserved in all
eukaryotes.
CHROMODOMAIN PROTEINS
Currently, there are over 100 identified and putative chromodomain proteins in the available databases. A review on mammalian chromodomain proteins was recently published (Jones et al., 2000). Here,
only aspects relevant for plant proteins will be
discussed.
Known chromodomain proteins may be grouped
into two classes, based on whether they are heterochromatin components or not (Table I).
Class A contains two Su(var) families. The HP1
(heterochromatin protein) family is represented by
about 20 members from different species. All are
products of genes homologous to the D. melanogaster
Su(var)2-5 gene. HP1 is a structural component of
heterochromatin and a dose-dependent modifier of
PEV. Therefore, HP1 is a major factor bridging gene
silencing with heterochromatin structure (for review,
see Eissenberg and Elgin, 2000).
A signature feature of the HP1 family proteins is
their relatively small size (171–328 amino acids) and
the presence of two motifs: a chromodomain, at the N
end, and a chromoshadow domain, at the C end. The
chromodomain and the chromoshadow domain are
about 60% similar to each other. They do not bind
DNA, are involved in protein/protein interactions,
and are needed for targeting HP1 to heterochromatin
(Pak et al., 1997; for a comprehensive list of factors
specifically binding HP1, see Wallrath, 1998; Eissenberg and Elgin, 2000; Jones et al., 2000).
No HP1 homolog may be found in the available
genome sequence of S. cerevisiae. In contrast, a gene
in fission yeast, Swi6, is 46% identical to Su(var)2-5
over its entire sequence. It is involved in carrying
epigenetic information through mitosis and meiosis
and in the assembly and propagation of heterochromatin (for review, see Jenuwein and Allis, 2001).
The second family of class A contains proteins from
different species encoded by homologs of the D. melanogaster Su(var)3-9 gene. The characteristic feature of
these proteins is that their chromodomain is in combination with a Su(var), E(z), Trithorax (SET) domain. The SET domain is a highly conserved, approximately 150-amino acid motif shared by a large
number of eukaryotic transcriptional activators and
repressors. The SET domain proteins of yeast, animal, and plant origin have been recently systematized and comprehensively analyzed (Baumbusch et
al., 2001; Alvarez-Venegas and Avramova, 2002) and
will not be discussed here. However, it is interesting
to note that there is a large number of SET domain
genes in Arabidopsis and that none of them is in
combination with a chromodomain. Apparently,
there is no homolog of the heterochromatin specific
Su(var)3-9 protein in Arabidopsis. There is no homolog of Su(var)3-9 in the budding yeast S. cerevisiae
as well, in contrast with the fission yeast. The fission
yeast Clr4 gene is a homolg of Su(var)3-9 (for review,
Table I. Classes of chromodomain proteins
⫹ and ⫺, Presence or absence of a particular motif; ⫹⫹, presence of two identical motifs. MOF, Male-absent On the First protein. MSL,
Male-Specific Lethal protein. CHD, Chromodomain, Helicase domain, DNA-binding domain protein. CAO, Chlorophyll a/b-binding harvesting
organelle-specific protein.
Family
Class A
HP1
Su(var)3-9
Class B
Polycomb
MOF
MSL
CHD
Methylases
CAO
LTR retroelements
42
Chromo
Chromoshadow
⫹
⫹
⫹
–
–
SET
Jones et al. (2000)
Jones et al. (2000)
⫹
⫹
⫹
⫹⫹
–
–
–
–
–
–
–
SNF2 helicase, Zn fingers,
bromo, PHD, and DNA
binding
Putative methyltransferase
active center
–
pol
Jones et al. (2000)
Jones et al. (2000)
Jones et al. (2000)
Woodage et al. (1997)
⫹
⫹⫹
⫹
–
Other Domains
Reference
Henikoff and Comai (1998)
Klimyuk et al. (1999)
Koonin et al. (1995); Malik and
Eickbush (1999); Z. Avramova
(unpublished data)
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Plant Physiol. Vol. 129, 2002
Heterochromatin in Animals and Plants
Figure 1. Models for heterochromatin formation in different organisms. A, S. cerevisiae. The tails of histone H4 contain
several lysines that undergo specific deacetylation. RAP1 factor and the four-subunit silence information regulator (SIR)
complex assemble on the deacetylated H4 tails. These complexes have a propensity to self propagate resulting in the densely
packed structure characteristic of heterochromatin. The origin of replication recognition complex (ORC), recruited through
its specific binding to SIR1, is involved also in the multimeric heterochromatic complexes. B, Human heterochromatin. The
complexes assemble on the histone H3 tails. Deacetylated Lys-9 serves as a substrate for the methylating activity of the SET
peptide of the SUVAR39H1 protein, a human homolog of the D. melanogaster Su(var)3-9. The methylated Lys provides a
binding site for the chromodomain of HP1. It binds and recruits new molecules of SUVAR39H1 providing a mechanism for
self-propagation. The specific binding of ORC1 subunit to HP1 may result in a similar multimeric complex as in yeast. C,
Fission yeast. The mechanism is very similar to that of human. Clr4 is the ortholog of SUVAR39H1, whereas Swi6 is an
ortholog of HP1. Two different deacetylases are involved in the specific deacetylation of the two lysines on the H3 tail. Note
also that only the chromomotifs of the HP1 family can bind methylated lysines.
see Jenuwein and Allis, 2001; Fig. 1). The roles of the
SET and the chromodomains in heterochromatin formation will be discussed later.
Plant Physiol. Vol. 129, 2002
Class B is heterogeneous, consisting of several
families. The unifying feature for this class is that
the proteins carry the chromodomain motif (in one
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43
Avramova
or more copies) in combination with various other
motifs, but not the chromoshadow or the SET
motifs.
The D. melanogaster Polycomb and two chromodomain-containing proteins with activating functions, MOF and MSL-3, belong to this class (Table I).
There are no S. cerevisiae or plant homologs for any of
the genes from these three families. However, multiple plant genes belong to a large superfamily of
chromodomain genes, the CHD superfamily (http://
chromdb.biosci.arizona.edu/). In S. cerevisiae, the single chromobox gene in the entire genome (L10718)
belongs in the CHD superfamily. Members of the
CHD superfamily are implicated in chromatin remodeling activities conserved in yeast and in higher
eukaryotes (Woodage et al., 1997).
A family of approximately 20 putative DNA methyltransferases, containing a chromobox in the putative active center, is unique for plants (Henikoff and
Comai, 1998). One of them, CMT3, has been implicated in the plant-specific methylation of CpXpG
(Lindroth et al., 2001). Chromomethyltransferases,
together with a chloroplast-specific gene containing
two chromoboxes (CAO; Klimyuk et al., 1999), are
fascinating examples of chromodomain proteins that
apparently have evolved for plant-specific functions.
Last, chromobox homologous motifs were found in
a retrovirus (Malik and Eickbush, 1999), in the Ty3
class of retrotransposons (Koonin et al., 1995) and in
Arabidopsis and maize (Tekay and Rle) retrotransposons. The two Arabidopsis (accession nos.
AAD39272 and AAF13073) and maize (accession nos.
AF050455 and AF057037) retrotransposons contain
chromomotifs in their pol genes that are 55%, 54%,
57%, and 49% similar to the human HP1 chromobox,
respectively (Z. Avramova, unpublished data). A
function for these motifs is not evident, but the structural conservation of the motif in thermophilic archaebacteria Sulfolobus acidocaldarius and S. solfataricus
(Ball et al., 1997) and in retroelements suggests that
the chromodomain is an ancient structural motif that
has acquired divergent functions in evolution.
In summary, with respect to heterochromatin,
classes A and B are distinguished by the fact that
only the members of class A are components of heterochromatin. Despite the presence of chromodomains in the proteins of class B, no involvement with
heterochromatin has been established for any of them
(see below and Fig. 1). Members of class B may
participate in both silencing and activation processes,
or even in processes unrelated to chromatin, as could
be the case with CAO.
Silencing Complexes in S. cerevisiae
A few loci in S. cerevisiae, the silent mating, ribosomal, and telomeric regions, can repress juxtaposed
genes through the formation of large multiprotein
complexes (for review, see Grunstein, 1998; Fig. 1A).
Attention is focused on the involvement of the tails of
histone H4 and of two complexes, the ORC and the
SIR complexes, in the assembly mechanism. A review
of ORC and SIR complexes is beyond the scope of
this paper and relevant reviews are recommended
(Grunstein, 1998; Lee and Bell, 2000).
Following deacetylation of histone H4 N-tail lysines, the SIR and ORC complexes bind specifically
the RAP1 protein on the protruding tails (Fig. 1A).
The specific interaction between Sir1 and Orc1 subunits is responsible for the recruitment of the remaining complexes to the sites of nucleation of extended
multiprotein complexes. Genes integrated in the vicinity of such complexes are silenced in a manner
similar to PEV in D. melanogaster (Wallrath, 1998). In
S. cerevisiae, therefore, despite the lack of morphologically distinct heterochromatin and the absence of
genes homologous to the D. melanogaster heterochromatin genes, large-scale gene silencing appears similar to PEV. On this basis, the silencing loci of yeast
are considered functional equivalents of heterochromatin (Huang et al., 1998).
Silencing Complexes in Higher Eukaryotes
Earlier models for the silencing activity of animal
heterochromatin were based on mechanisms commanding long-range gene silencing in yeast. The role
of protein methylases in the long-standing mystery
of heterochromatin is addressed in a recent review
(Jenuwein and Allis, 2001; Fig. 1B).
Two major distinctions in the formation of heterochromatin and the yeast silencing complexes are the
involvement of the tails of histone H3, instead of H4,
and the fact that the assembly of the multimeric
complexes is preceded by a specific methylation of
histone H3-Lys-9. The enzyme responsible for the
methylating reaction, SUV39H1, is the product of the
human homolog of the D. melanogaster Su(var)3-9
(Rea et al., 2000). Methylated His-3-Lys-9 creates a
specific binding site for the chromodomain of the
human homolog of HP1 (Bannister et al., 2001; Lachner et al., 2001). Bound HP1 could recruit new
SUV39H1 molecules to methylate other histones after
replication, providing a mechanism for heterochromatin formation and propagation (Jenuwein and Allis, 2001).
HOW DOES HETEROCHROMATIN SILENCE
GENES?
Silencing Complexes in Fission Yeast
Formation of extended multimeric protein complexes is the basis of heterochromatin formation. It
has been postulated that it also may provide a basis
of heterochromatin silencing (see below).
In fission yeast, the products of two genes, Clr4 and
Swi6, homologs of Su(var)3-9 and Su(var)2-5, respectively, are involved in H3-Lys-9 modification and
subsequent heterochromatin formation in a manner
44
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Plant Physiol. Vol. 129, 2002
Heterochromatin in Animals and Plants
similar to that in animals (Nakayama et al., 2001). An
important detail in the mechanism is the identification of a specific histone H3-Lys-14 deacetylase, Clr3.
Following H3-Lys-9 and Lys-14 deacetylation, Clr4 is
recruited to methylate H3-Lys-9. Swi6 binds subsequently (Fig. 1C).
The Histone Code
Alteration of the nucleosomal structure via histone
modificationis is a major principle upon which current models of heterochromatin formation are built.
Because Lys-9 in H3 can be either acetylated or methylated, competitive modification at this position provides a molecular switch for induction of hetero- or
euchromatic subdomains. This coordinate mode of
chemical modification of the core histone tails is the
basis of the histone code hypothesis (for review, see
Jenuwein and Allis, 2001). Histone amino-terminal
modifications create or destroy affinities for other
chromatin-binding proteins. This, in turn, commands
transitions between active and inactive states. The
combinatorial nature of the modifications reveals a
“histone code” that extends the informational potential of genetic code. The histone code, therefore, represents an epigenetic mark and a regulatory mechanism (Jenuwein and Allis, 2001).
What about Plants?
The important discovery of histone H3-methylase
activity of the SET-domain of the human SUV39H1
protein was triggered by an observed weak sequence
homology with plant protein methyltransferases (Rea
et al., 2000). Six homologous plant sequences are
classified as potential histone Lys transferases but
only one has been functionally characterized and
found to lack histone methylase activity (Klein and
Houtz, 1995). Therefore, the question of whether the
Lys tails of histone H3 undergo specific methylation
in plant heterochromatin assembly remains open (see
“Note Added in Proof”). Another question is the
absence in the Arabidopsis genome of a sequence
homolog of Su(var)3-9, suggesting that if H3-Lys
methylation does take place in plant heterochromatin, it is accomplished by a protein that is not a
sequence homolog of the animal Su(var)3-9 proteins.
Furthermore, if such methylation does take place
during heterochromatin assembly, a compelling
question is which protein, if any, would bind to the
methylated lysines to induce formation of extended
complexes.
The possibility that homologs of the animal heterochromatin genes still might be found in Arabidopsis
could be kept open until the entire genome is finished (gaps in a few centromeric regions have not
been filled out yet). It is interesting that at least 10
hypothetical proteins carrying an SET domain related to the SET of Su(var)3-9 may be found in the
Plant Physiol. Vol. 129, 2002
Arabidopsis database (Baumbusch et al., 2001;
Alvarez-Venegas and Avramova, 2002). None of
them contains a chromodomain and methylase activity has not been shown for their SET domains, but
these putative proteins could be potential histonemethylases and plant heterochromatin components.
Another possible candidate could be the ORC, involved in DNA replication and in silencing in both
yeast and D. melanogaster.
Connection between Heterochromatin, Silencing, and
ORC in Higher Eukaryotes
The involvement of the ORC in heterochromatin
formation in S. cerevisiae is illustrated in Figure 1.
The specific interactions of the yeast Sir1 with Orc1
and of the D. melanogaster HP1 with ORC1 provide an
important parallel in the formation of silencing complexes in the two species. The ORC is a dosagesensitive modifier of PEV. ORC1 subunit binds both
the chromo- and chromoshadow domains of HP1
and is essential for targeting HP1 to heterochromatin.
The yeast SIR1 and HP1 are considered functional
homologs, despite the lack of sequence similarity between the two genes (Pak et al., 1997; Huang et al.,
1998). The possibility that plants may have heterochromatin-silencing complexes, in the absence of gene
homologs of heterochromatin proteins, has its major
argument in this analogy.
Genes homologous to the six subunits of the yeast
ORC were discovered in various eukaryotes, including humans. A rice (Oryza sativa) homolog of ORC1
and an Arabidopsis homolog of ORC2 were reported
(Gavin et al., 1995; Kimura et al., 2000). The finding of
plant ORC subunits makes it plausible that plant
factors capable to bind the ORC might exist. If such
factors were found and shown to nucleate formation
of extended silencing complexes, they could be considered functionally equivalent to Sir1/HP1. However, it is not established yet whether an ORC exists
and functions in plants. Evidently, a study of plant
heterochromatin will inevitably raise questions about
a role of a putative plant ORC and its subunits.
IS SILENCING BY THE PC-G FACTORS
CONNECTED TO HETEROCHROMATIN?
A paradigm for an epigenetic silencing mechanism
is the repression of the homeotic genes. The propagation throughout development of the silenced gene
patterns established early in embryogenesis is
achieved by a set of proteins belonging to the Pc-G
(for review, see Cavali and Paro, 1998).
Relevant for our discussion are two members of the
Pc-G, products of the Polycomb and E(z) genes, because Polycomb carries a chromodomain, whereas
E(z) caries a SET domain. Because these motifs were
found also in the two heterochromatin proteins HP1
and Su(var)3-9, it was proposed that the silencing
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Avramova
potential of Polycomb and E(z) might be contained in
their ability to trigger heterochromatin assembly. It
sounded plausible and provided an attractive model
linking repression at euchromatic loci (the homeotic
genes) with a PEV-type silencing (for review, see
Cavali and Paro, 1998). The presence of similar architectural motifs between the Su(var)2-5 and
Su(var)3-9 proteins and the Pc-G members was considered the molecular link connecting the two types
of silencing activities.
However, as recently shown, the SET domain of the
homeotic gene regulator E(z) does not have a histone
H3-Lys-9 methylase activity, and only the chromodomains of Su(var)2-5 homologs, but not those of
class B proteins, could bind the methylated Lys-9
initiating heterochromatin formation (for review, see
Jenuwein and Allis, 2001). The different biochemical
activities of the SET domain of a heterochromatin
protein and the SET domain of a Pc-G protein, together with the different activities of the chromodomains from a Pc-G and from heterochromatin proteins, compellingly suggests that the model needs to
be revisited.
In summary, finding of similar peptide motifs does
not prove involvement in similar type functions.
Gene repression by the Pc-G proteins and formation
of heterochromatin probably involve different molecular mechanisms. Gene silencing mechanisms should
not be indiscriminately linked to PEV.
EVOLUTION OF HETEROCHROMATIN FUNCTION
In evolution, the origin of heterochromatin appears
to have paralleled the increase in size of some eukaryotic genomes. The densely compacted regions
may have occurred from the need to accommodate
excess amounts of predominantly foreign DNA. Because large invasions by mobile elements could be
deleterious for the hosts, cells have evolved different
ways to cope with this DNA by modifying it, silencing it, making it recombination deficient, and segregating it into gene-poor compartments defined as
heterochromatin. Eventually, cells may have taken
advantage of the presence of heterochromatin in their
nuclei, recruiting it for functions at the telomeres and
centromeres, for the correct folding and segregation
of mitotic chromosomes, and for the pairing and
synapsis of homologous chromosomes (Bass et al.,
2000). These functions have co-evolved with specifically interacting proteins, giving rise to the characteristic DNA/protein complexes. Then, it would be
the structure of the entire complex and the physical
characteristics associated with it, not the sequences
per se, that could be a primary determinant of function. This is compatible with the idea of the epigenetic
nature of heterochromatin formations (for review, see
46
Henikoff et al., 2001; Martienssen and Colot, 2001). It
is not known what the structure of plant heterochromatin could be, whether plant heterochromatin possesses gene-silencing potential, and whether it has
evolved as a global epigenetic regulatory mechanism
in plants.
Is There PEV in Plants?
PEV effects have not been studied systematically in
plants. In two early studies, variegated gene expression in Oenothera blandina has been reported following x-ray chromosomal disruptions and translocations (Catcheside, 1938; 1949). Although proximity to
heterochromatin was not demonstrated, it was suggested that the mosaic expression observed at the P
locus could be analogous to the PEV effects described
in D. melanogaster. An important conclusion from
these early studies was that the factor responsible for
the altered expression was the dislocation of the gene
from its natural position.
In only one study has a relationship between unstable transgene expression and pericentromeric insertion been reported in plants (Iglesias et al., 1997).
Despite the lack of experimental data, failures to
achieve expression of transgenes are routinely attributed to PEV (Matzke and Matzke, 1998).
If blocks of highly repetitive methylated DNAs
underlie the formation of plant heterochromatin, one
may expect that genes in and around such regions
would be silenced. However, recent genome studies
provided data that are difficult to reconcile with this
notion. Thus, in the adh 1 region of maize, solitary
genes exist among blocks of highly repetitive, methylated retrotransposons (SanMiguel et al., 1996; Tikhonov et al., 1999). Notably, in the colinear sorghum
(Sorghum bicolor) region, there are no such blocks in
the space between the genes (Tikhonov et al., 1999).
Likewise, extended regions of repetitive DNAs in the
maize Sh2-A1 region provide a different chromosomal milieu for the maize genes than for their homologs in sorghum and rice (Chen et al., 1998). Given
the presumed importance of the genomic context for
the correct function of a gene, these comparative
studies of monocots point to an apparent paradox:
Orthologous genes in related species function in substantially different chromosomal environments.
Several possibilities could be considered. One is
that plant heterochromatin is a structural feature
only (accommodating excess amounts of mobile
DNAs) and does not possess silencing capacity. Another is that plant genes residing among large blocks
of repetitive methylated DNAs resemble the D. melanogaster heterochromatin-specific genes. A few genes
in D. melanogaster (including the essential rolled and
light) are an interesting exception to the norm. They
reside and function within heterochromatin, are
dominantly silenced by mutations in Su(var)2-5, and
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Plant Physiol. Vol. 129, 2002
Heterochromatin in Animals and Plants
require heterochromatin for their expression (Lu et
al., 2000, and refs. therein). These heterochromatinspecific genes, therefore, ought to be clearly distinguished from the other genes because their expression
depends upon factors present in heterochromatin and
not upon mechanisms counteracting its silencing powers. Genes with similar expression requirements have
not been reported in other species and there are no
data to support such a possibility in plants.
Alternatively, plant heterochromatin may possess
gene-silencing potential (albeit the nature of the proteins forming the complexes is not discovered yet),
but the repressive activity has co-evolved with mechanisms protecting nearby genes from silencing.
Escaping Silencing by Heterochromatin
If one assumes that plant heterochromatin has a
repressive capacity, then it would be necessary to
reconcile facts of the presence of functional genes
among repetitive retroelements and, maybe, even inside pericentromeric and knob heterochromatin
(Fransz et al., 2000; McCombie et al., 2000). Current
models are based on ideas that genes in native systems have evolved mechanisms to protect their function at their natural locations and that mislocation
alters expression. These models suggest that genes
and blocks of highly repetitive DNAs exist in separate structural domains or nuclear compartments (for
review, see Lamond and Earnshaw, 1998). Each gene
in the nucleus may have only one “address” at which
it functions correctly and during evolution, genes
have acquired “anchors” to position them stably in
the spatial architecture of the nucleus. A specific class
of DNA sequences, matrix attachment regions
(MARs) may be involved in this anchoring function.
The genes in the adh1 region of maize and in the
sh2-a1 regions of rice and sorghum might be segregated into putative structural loops, separated from
neighboring genes, non-genic sequences, and long
blocks of repetitive elements (Avramova et al., 1995,
1998; Tikhonov et al., 2000). Despite the tendency of
retroelements to insert into older retroelements (SanMiguel et al., 1996), it is significant that the initial
retrotransposons, those found at the base of the stack
map right at, or in a very close proximity to, MARflanking genes (Tikhonov et al., 2000). In addition, a
class of non-long terminal repeat (LTR) retrotransposable elements, short interspersed nuclear elements, preferentially target regions in the genome of
Brassica that display MAR characteristics (Tikhonov
et al., 2001). These results suggest that in plants,
MARs might act both as potential target sites and as
barriers for the genes against deleterious invasion by
LTR and non-LTR retrotransposons.
An exciting possibility for a molecular characterization of the borders between hetero- and euchromatin is provided by the recently sequenced regions
Plant Physiol. Vol. 129, 2002
expanding over the morphological boundaries of heterochromatin (McCombie et al., 2000).
Plant Heterochromatin and Evolution of
Silencing Mechanisms
A tantalizing question is whether plants, in their
evolution, have made use of heterochromatin in
large-scale silencing mechanisms like animals have.
An answer to this question may have important evolutionary implications. According to conventional
theories, plants and animals have diverged from a
unicellular ancestor (Baldauf and Palmer, 1993). Separation from a unicellular ancestor would indicate
that plants and animals have independently achieved
multicellularity and the mechanisms regulating it. It
may be expected, therefore, that different principles
(genes) would govern the balance between proliferation/differentiation and homeotic gene regulation
because in plants, organ development is not restricted to the embryonic stage and organogenesis/
differentiation occurs throughout the life span. This
could suggest that plants and animals have also differently evolved their heterochromatin, using it as a
global silencing factor in animals but not in plants.
The conservation of the yeast ORC/Sir1 and the D.
melanogaster ORC/HP1 interactions suggests that, in
their evolution to multicellularity, animals may have
inherited principles and mechanisms for large-scale
silencing from a unicellular predecessor. These
mechanisms have been modified respectively to suite
animal-specific needs. We may ask whether plant
heterochromatin has also adapted a silencing principle from a unicellular ancestor and evolved it for
plant-specific functions.
These fascinating possibilities have not been explored yet. It is possible that common principles will
be revealed for animals and plants despite differences in their developmental and survival strategies.
It is also evident that mechanisms unique for plants
will be revealed that will illustrate the diversity of
scenarios played by nature in its evolution to multicellular organisms. Studying plant heterochromatin
provides such an opportunity.
Note Added in Proof
Recent groundbreaking results in Neurospora sp. and
Arabidopsis provided evidence for a connection between
DNA methylation and histone H3 K9 methylation (H.
Tamaru, E.U. Selker [2001] Nature 414: 277–283; J.P. Jackson, A.M. Lindroth, X. Cao, S.E. Jacobsen [2002] Nature
416: 556–560). In addition, the latter paper provided first
evidence for the existence of histone H3 K9 methylation in
plants, for the activity responsible for this modification,
and for its connection to plant-specific CpNpG DNA methylation. A newly reported HP1-like factor from Arabidopsis (V. Gaudin, M. Libault, S. Poteau, T. Juul, G. Zhao, D.
Lefebre, O. Grandjean [2001] Development 128: 4847–4858)
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47
Avramova
is involved in mediating the control of CpNpG DNA methylation by H3 K9 methylation (Jackson et al., 2002). Collectively, these new results transform one of the differences
between animals and plants, i.e. absence of reported plant
heterochromatin proteins, into a similarity and provide
answers to some of the most compelling questions raised
earlier in the review.
ACKNOWLEDGMENTS
I am grateful to James Birchler (University of Missouri,
Columbus), to Steve Henikoff (Fred Hutchison Center for
Cancer Research, Seattle), and to Rob Martienssen (Cold
Spring Harbor Laboratory, New York) for their critical
reading, helpful comments, and suggestions on the manuscript. Jane Einstein is gratefully acknowledged for the
preparation of Figure 1. I apologize to all colleagues whose
works were not cited because of space limitations. Nonetheless, their published results have shaped my understanding of heterochromatin and have provided a basis for
this review.
Received October 29, 2001; accepted January 25, 2002.
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