Plant Physiol, May 2002, Vol. 129, pp. 40-49

UPDATE ON HETEROCHROMATIN
Heterochromatin in Animals and Plants. Similarities and Differences

School of Biological Sciences, Manter Hall, University of Nebraska, Lincoln, Nebraska 68588

	INTRODUCTION

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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).

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 eukaryotic 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

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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 epigenetic 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)- 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

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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).

View this table: [in this window] [in a new window]   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.

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, see Jenuwein and Allis, 2001; Fig. 1). The roles of the SET and the chromodomains in heterochromatin formation will be discussed later.

View larger version (32K): [in this window] [in a new window]   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.

Class B is heterogeneous, consisting of several families. The unifying feature for this class is that the proteins carry the chromodomain motif (in one 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.

	HOW DOES HETEROCHROMATIN SILENCE GENES?

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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).

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).

Silencing Complexes in Fission Yeast

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 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 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 histone-methylases 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 dosage-sensitive 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?

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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 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

TOP INTRODUCTION MOLECULAR COMPONENTS OF... CHROMODOMAIN PROTEINS HOW DOES HETEROCHROMATIN... IS SILENCING BY THE... EVOLUTION OF HETEROCHROMATIN... LITERATURE CITED

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 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 require heterochromatin for their expression (Lu et al., 2000, and refs. therein). These heterochromatin-specific 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, MAR-flanking 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 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) 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.

	FOOTNOTES

Received October 29, 2001; accepted January 25, 2002.

^* E-mail zavramova2{at}unl.edu; fax 402-472-2083.

www.plantphysiol.org/cgi/doi/10.1104/pp.010981.

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