Heterochromatin in Animals and Plants. Similarities and Differences

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 ofDrosophila 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 inSaccharomyces 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. 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 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. 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 (TableI). Classes of chromodomain proteins + and −, Presence or absence of a particular motif; ++, presence of two identical motifs. MOF, Male-absent On theFirst protein. MSL, Male-SpecificLethal protein. CHD, Chromodomain,Helicase domain, DNA-binding domain protein. CAO,Chlorophyll a/b-binding harvestingorganelle-specific protein. Classes of chromodomain proteins + and −, Presence or absence of a particular motif; ++, presence of two identical motifs. MOF, Male-absent On theFirst protein. MSL, Male-SpecificLethal protein. CHD, Chromodomain,Helicase domain, DNA-binding domain protein. CAO,Chlorophyll a/b-binding harvestingorganelle-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 similar to They not DNA, are involved in and are for HP1 to heterochromatin et al., for a of see Eissenberg and Elgin, 2000; et al., 2000). No HP1 may be found in the available cerevisiae. In a gene in fission yeast, is identical to Su(var)2-5 over its It is involved in epigenetic information and and in the assembly and propagation of heterochromatin (for review, see and 2001). for heterochromatin formation in different S. cerevisiae. The of histone contain several that specific factor and the silence information complex on the deacetylated These have a to in the densely packed structure of heterochromatin. The of complex its specific to is involved also in the heterochromatin. The on the histone as a for the activity of the of the a of the D. The methylated a for the chromodomain of It and of a mechanism for The specific of to HP1 may result in a similar complex as in The mechanism is similar to that of is the of whereas is an of different deacetylases are involved in the specific deacetylation of the two on the also that only the of the HP1 family can methylated Class B is of several families. The feature for is that the proteins the chromodomain one or more in with other but not the chromoshadow or the motifs. The D. melanogaster Polycomb and two proteins with and belong to There are cerevisiae or plant homologs for any of the genes from these three families. However, plant genes belong to a large superfamily of chromodomain genes, the superfamily In S. the gene in the in the of the superfamily are implicated in chromatin activities conserved in yeast and in et al., A family of 20 putative DNA containing a in the putative active is unique for plants (Henikoff and of has been implicated in the plant-specific methylation of et al., 2001). with a gene containing two et al., are fascinating of chromodomain proteins that have evolved for plant-specific homologous were found in a and in the of et al., and in Arabidopsis and maize The two Arabidopsis and and maize contain in their that are and similar to the HP1 A function for these is not but the structural of the in S. et al., and in that the chromodomain is an structural that has functions in In with to heterochromatin, A and B are by the fact that only the members of A are components of heterochromatin. Despite the presence of in the proteins of involvement with heterochromatin has been established for any of them (see and of B may in both silencing and or even in processes to as be the with of extended protein is the basis of heterochromatin formation. It has been that it also may provide a basis of heterochromatin silencing (see below). A loci in S. the and regions, can genes the formation of large (for review, is on the involvement of the of histone and of two the and the in the assembly mechanism. A review of and is beyond the scope of and relevant reviews are recommended and 2000). deacetylation of histone the and the protein on the The specific and is responsible for the of the to the of of extended Genes in the vicinity of such are silenced in a similar to PEV in D. melanogaster In S. despite the lack of heterochromatin and the absence of genes homologous to melanogaster heterochromatin genes, large-scale gene silencing appears similar to PEV. On the silencing loci of yeast are of heterochromatin et al., for the silencing activity of animal heterochromatin were based on mechanisms gene silencing in The role of protein in the of heterochromatin is in a recent review and 2001; major in the formation of heterochromatin and the yeast silencing are the involvement of the of histone of and the fact that the assembly of the is by a specific methylation of histone The responsible for the is the of the of the D. melanogaster Su(var)3-9 et al., 2000). a specific for the chromodomain of the of HP1 et al., 2001; et al., 2001). HP1 recruit to other histones a mechanism for heterochromatin formation and propagation and 2001). In fission yeast, the products of two genes, homologs of Su(var)3-9 are involved in and heterochromatin formation in a similar to that in animals et al., 2001). An important detail in the mechanism is the identification of a specific histone and is to of the structure histone is a major current of heterochromatin formation are in can be either or methylated, at position a molecular for of or euchromatic This of of the histone is the basis of the histone (for review, see and 2001). modifications or for other in active and inactive The nature of the modifications a that the potential of genetic The histone an epigenetic and a regulatory mechanism and 2001). The important discovery of histone activity of the of the protein was by an with plant protein methyltransferases et al., 2000). homologous plant sequences are as potential histone but only one has been functionally and found to lack histone activity and Therefore, the of whether the of histone specific methylation in plant heterochromatin assembly remains (see “Note Added in is the absence in the Arabidopsis of a suggesting that methylation does in plant heterochromatin, it is by a protein that is not a of the animal Su(var)3-9 such methylation does heterochromatin a is would to the methylated to formation of extended The that homologs of the animal heterochromatin genes be found in Arabidopsis be kept the is in a centromeric regions have not been filled It is that at least proteins an domain related to the of Su(var)3-9 may be found in the Arabidopsis et al., and of them contains a chromodomain and activity has not been for their but these putative proteins be potential and plant heterochromatin possible be the involved in DNA and in silencing in both yeast and D. The involvement of the in heterochromatin formation in S. cerevisiae is in The specific of the yeast with and of melanogaster HP1 with provide an important in the formation of silencing in the two species. The is a modifier of PEV. both the and chromoshadow of HP1 and is essential for HP1 to heterochromatin. The yeast and HP1 are despite the lack of the two genes et al., et al., The that plants may have in the absence of gene homologs of heterochromatin has its major in Genes homologous to the of the yeast were discovered in including A of and an Arabidopsis of were et al., et al., 2000). The of plant it that plant to the such were found and to formation of extended silencing they be functionally to However, it is not established yet whether an and functions in a of plant heterochromatin will raise questions about a role of a putative plant and its A paradigm for an epigenetic silencing mechanism is the repression of the genes. The propagation throughout of the silenced gene established in is by a of proteins to the (for review, and for our are two members of the products of the Polycomb and genes, because Polycomb a chromodomain, whereas a these were found also in the two heterochromatin proteins HP1 and it was that the silencing potential of Polycomb and be in their to heterochromatin It and provided an attractive repression at euchromatic loci with a silencing (for review, see and The presence of similar the Su(var)2-5 and Su(var)3-9 proteins and the members was the molecular link the two types of silencing However, as recently the domain of the gene does not have a histone and only the of Su(var)2-5 but not those of B the methylated heterochromatin formation (for review, see and 2001). The different biochemical activities of the domain of a heterochromatin protein and the domain of a with the different activities of the from a and from heterochromatin that the to be In of similar does not involvement in similar Gene repression by the proteins and formation of heterochromatin probably different molecular Gene silencing mechanisms not be to PEV. In the of heterochromatin appears to have the in size of some eukaryotic The densely regions may have from the to of large by mobile be for the have evolved different to with DNA by silencing it and it into defined as heterochromatin. may have of the presence of heterochromatin in their it for functions at the and centromeres, for the correct folding and of and for the and of homologous chromosomes et al., 2000). These functions have with to the it would be the structure of the complex and the associated with not the sequences that be a of function. This is with the of the epigenetic nature of heterochromatin (for review, et al., 2001; Martienssen and Colot, 2001). It is not known what the structure of plant heterochromatin whether plant heterochromatin and whether it has evolved as a global epigenetic regulatory mechanism in PEV effects have not been in In two gene expression in has been following chromosomal and Although to heterochromatin was not it was that the mosaic expression at the be to the PEV effects in D. An important from these studies was that the factor responsible for the expression was the of the gene from its In only one has a expression and pericentromeric been in plants et al., Despite the lack of to expression of are to PEV and Matzke, of highly repetitive methylated DNAs underlie the formation of plant heterochromatin, one may that genes in and such regions would be However, recent studies provided that are to with Thus, in of genes among of highly repetitive, methylated et al., et al., 1999). in the colinear there are such in the the genes et al., 1999). extended regions of repetitive DNAs in the maize provide a different chromosomal for the maize genes than for their homologs in and et al., the of the context for the correct function of a these studies of to an genes in related species function in different chromosomal be is that plant heterochromatin is a structural feature only of mobile and does not silencing is that plant genes among large of repetitive methylated DNAs the D. genes. A genes in D. melanogaster (including the essential are an to the They and function heterochromatin, are silenced by in and heterochromatin for their expression et al., and refs. therein). These genes, to be from the other genes because their expression in heterochromatin and not mechanisms its silencing Genes with similar expression have not been in other species and there are to such a in plant heterochromatin may potential the nature of the proteins the is not discovered but the repressive activity has with mechanisms nearby genes from one that plant heterochromatin has a repressive it would be to of the presence of genes among repetitive even pericentromeric and knob heterochromatin (Fransz et al., 2000;McCombie et al., 2000). are based on that genes in have evolved mechanisms to their function at their and that These that genes and of highly repetitive DNAs in structural or nuclear (for review, and gene in the nucleus may have only one at it functions and genes have to position them in the of the A specific of DNA sequences, regions may be involved in function. The genes in the of maize and in regions of and be into putative structural from genes, sequences, and of repetitive et al., et al., 2000). Despite the of to into et al., it is that the those found at the of the or in a genes et al., 2000). In addition, a of nuclear target regions in the of that display et al., 2001). These results that in plants, both as potential target and as for the genes by and An for a molecular of the and euchromatin is provided by the recently sequenced regions over the morphological of heterochromatin et al., 2000). A is whether plants, in their have made of heterochromatin in large-scale silencing mechanisms like animals have. An to may have important According to conventional plants and animals have from a and from a would that plants and animals have and the mechanisms It may be that different would the and gene because in plants, is not to the and throughout the This that plants and animals have also evolved their heterochromatin, it as a global silencing factor in animals but not in The of the yeast and the D. melanogaster in their to animals may have and mechanisms for large-scale silencing from a These mechanisms have been to may whether plant heterochromatin has also a silencing from a and evolved it for plant-specific These fascinating have not been It is possible that will be for animals and plants despite differences in their and It is also that mechanisms unique for plants will be that will the of by nature in its to plant heterochromatin such an Recent results in and Arabidopsis provided for a DNA methylation and histone methylation In addition, the latter provided first for the of histone methylation in plants, for the activity responsible for and for its to plant-specific DNA A factor from Arabidopsis S. D. is involved in the of DNA methylation by methylation et al., these results one of the differences animals and plants, absence of plant heterochromatin into a and provide answers to some of the most questions in the I to of to Henikoff for and to Martienssen for their and on the is for the of I to all were not because of their published results have understanding of heterochromatin and have provided a basis for