Russian Journal of Genetics, Vol. 39, No. 2, 2003, pp. 133–146. Translated from Genetika, Vol. 39, No. 2, 2003, pp. 187–201. Original Russian Text Copyright © 2003 by Zhimulev, Belyaeva.
Heterochromatin, Position Effect, and Genetic Silencing I. F. Zhimulev1, 2 and E. S. Belyaeva1 1
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Institute of Cytology and Genetics, Russian Academy of Sciences, Novosibirsk, 630090 Russia Department of Cytology and Genetics, Novosibirsk State University, Novosibirsk, 630090 Russia; fax: (3832)30-16-65; e-mail:
[email protected] Received October 16, 2002
Abstract—Genomes of higher eukaryotes consist of two types of chromatin: euchromatin and heterochromatin. Heterochromatin is densely packed material typically localized in telomeric and pericentric chromosome regions. Euchromatin transferred by chromosome rearrangements in the vicinity of heterochromatin is inactivated and acquires morphological properties of heterochromatin in the case of position effect variegation. One of the X chromosomes in mammal females and all paternal chromosome set in coccides become heterochromatic. The heterochromatic elements of the genome exhibit similar structural properties: genetic inactivation, compaction, late DNA replication at the S stage, and underrepresentation in somatic cells. The genetic inactivation and heterochromatin assembly are underlain by a specific genetic mechanism, silencing, which includes DNA methylation and posttranslational histone modification provided by the complex of nonhistone proteins. The state of silencing is inherited in cell generations. The same molecular mechanisms of silencing shared by all types of heterochromatic regions, be it unique or highly repetitive sequences, suggest the similar organization of these regions. No type of heterochromatin is a permanent structure as they all are formed at the strictly definite stages of early embryogenesis. Based on the bulk of evidence accumulated today, heterochromatin can be regarded as a morphological manifestation of genetic silencing.
INTRODUCTION In the late 1920s–early 1930s, Heitz has shown that intensely stained bodies and grains found in interphase nuclei are either specific chromosomes or chromosome fragments maintaining compact state throughout the cell cycle. These structures were named heterochromatin while the remaining chromatin was termed euchromatin (true chromatin) [1, 2]. The subsequent discoveries and the advent of novel techniques permitted more precise determination of the notion of heterochromatin and its localization in chromosomes and genomes. New heterochromatin properties were revealed, such as inactive state of genes, late DNA replication, underrepresentation of DNA in somatic cells. Muller [3] showed that euchromatic genes are inactivated when transferred to the vicinity of pericentric heterochromatin—the phenomenon, which was later termed position effect variagation (PEV). These phenomena have been currently extensively studied and are the subject of numerous reviews [4–11]. The level of gene inactivation by PEV can be substatially modified by various factors [4, 12, 13]. The data on the heterochromatic inactivation of heterochromatin-adjacent genes provided researchers with a powerful tool for studying molecular mechanisms of the formation and preservation of epigenetically inherited inactivation of genetic material during individual development. The compact chromosome state in heterochromatic regions results from genetic inactivation referred to as silencing. This term has been coined in the mid-1970s
(see [7]) and was first used to denote constant transcription inactivation in the MAT locus of yeast Saccharomyces cerevisiae. Later it was extended to other cases of stable epigenetic gene inactivation. A role of several proteins in establishing silencing in heterochromatin has been revealed at the molecular level. In addition to silencing cytologically manifested by the heterochromatin formation, the mechanism of silencing is known that is provided by the Polycomb group (PcG) proteins for regulating the expression of homeotic genes BX-C and ANT-C during normal development of Drosophila [14–16]. The PcG proteins are partly homologous to the HP1 protein or its homologs localized in heterochromatic regions of many organisms [17]. Since cytological characteristics of chromosomes from the numerous localization regions of the PcG-associated genes have not been described so far, PcG silencing is not considered in this review. The main aim of the review is analysis of general characteristics of various phenomena related to genetic silencing: heterochromatin, position effect variegation, X-chromosome inactivation in mammalian females, and inactivation of the paternal chromosome set in coccides. TYPES OF HETEROCHROMATIN In the genomes of higher eukaryotes, heterochromatin can be represented by the total chromosome set of one of the parents, individual chromosomes (e.g., one
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Fig. 1. Correlations in the localization of Q-staining (shown in balck) detecting AT-rich heterochromatin regions (a) and late DNA replication (3H-thymidine inclusion) (b) in chromosomes of a metaphase plate of Samoaia leonensis. The Y chromosome and point chromosomes (thick arrow) are totally heterochromatic; autosomes show pericentric localization of heterochromatin; the X chromosome has one arm consisting of heterochromatin (Xh), a pericentric fragment (arrow), and another heterochromatic fragment in the euchromatic arm (Xe) (after [18]).
of the X chromosomes of females in mammals or the Y chromosome in Drosophila), supernumerary B and E chromosomes as well as separate chromosome segments (Fig. 1). The heterochromatin amount of the genome vary in a very wide range (see [6–8, 19–21 for review). In yeast, heterochromatin cannot be identified cytologically because of the small chromosome size but particular chromosome regions have many characteristics of heterochromatin [review in [22]). Pericentric heterochromatin and position effect variegation. Pericentric heterochromatin blocks are revealed in most plant and animal species examined as dense, specifically stained material (C-banding in metaphase chromosomes). Its amount varies in different species. Q- and H-banding of heterochromatin are also known (Fig. 1). In polytene chromosomes of Drosophila, pericentric heterochromatin blocks fuse to form a single chromocenter, in which very dense α-heterochromatin grains and more loose β-heterochromatin are detected (reviews in [6, 23]). Pericentric heterochromatin contains few genes and is represented by high and midrepetitive sequences. For instance, sequencing has shown that the euchromatic part of the Drosophila melanogaster genome contains approximately 13 000 genes [24] whereas only about 50 genes affecting viability and (or) fertility have been found in pericentric hetero-
chromatin which constitutes 30% of the genome. About 100 genes more are predicted in these regions on the basis of additional analysis of sequence data [10, 25]. Thus, the gene density in heterochromatin is 100 times lower than in euchromatin. Similar conclusions were made earlier [26] from cytogenetic data. A characteristic property of pericentric heterochromatin is the presence of long tracts, various satellite repeats whose length vary in different Drosophila species from 5–7 to 359 bp. Pericentric heterochromatin also contains many mid-repetitive sequences, which are often represented by mobile elements. Stable suppression of transcription in pericentric heterochromatin does not involve heterochromatic genes. Their specific regulation permit them to express normally in the neighborhood of silent heterochromatin (see [6, 7, 9] for review). One of the key characteristics of pericentric heterochromatin is its ability to expand its inactive state to euchromatic regions transferred in its vicinity via chromosome rearrangements. This phenomenon is known as position effect variegation (PEV). Variegation of inactivation of the transferred euchromatic genes is determined by cell-to-cell variation of the suppression effect in pericentric heterochromatin. Probably, genetic inactivation upon PEV spreads from the euchromatin– heterochromatin boundary as a result of the disruption
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of a barrier that normally limits the regions of heterochromatic silencing [27]. The heterochromatin inactivation upon PEV is accompanied by morphological alterations that make it similar to heterochromatin. This process of heterochromatization has been studied in detail in salivary gland polytene chromosomes of Drosophila. The transferred euchromatic region loses its characteristic banding pattern being transformed into blocks of compact material. This process sometimes involves very long regions containing more than 170 bands (over 5000 kb [28]). The genetic material involved in PEV compaction, becomes inaccessible for transcription factors. This was shown, for example, for the Broad-Complex gene, which is normally activated by hormone ecdysteron [29, 30]. Inactivation spreading subsides as the distance from heterochromatin increases; i.e., genes located close to the breakpoint are suppressed more often that the more remote ones. However, for some rearrangements heterochromatization was shown to be discontinuous: the compact zones alternate with the regions having normal banding (euchromatic) structure that retain their transcription activity [30]. Discontinuous compaction indicate different sensitivity of particular chromosome regions to the inactivating heterochromatin effect. A significant characteristic of PEV is its susceptibility to physical (e.g., temperature of embryonic development) and genetic (effects of additional heterochromatin and mutations in genes modifying PEV) (see [6] for details). The compact state of pericentric heterochromatin is not permanent [31], its appearance is strictly programmed and associated with a particular stage of development. The events of the formation (“maturation”) of heterochromatin in ontogeny are studied in detail in Drosophila. As early as in 1933, Huettner showed that in D. melanogaster, heterochromatin is not revealed in early embryonic development. This work as well as in subsequent electron-microscopic studies demonstrated that in the interphase nuclei of the first 11 to 12 cleavage divisions, the chromosome material is represented by a fine dispersed network without differentiation in eu- and heterochromatin. The heteropicnic chromocenter is detected only at the blastoderm stage (see [6] for references). C-banded heterochromatin was not also found in metaphase chromosomes at the early cleavage stages, it appears only after cell cycles 11–13 [32]. At the same time, specific heterochromatic proteins determining heterochromatic domain splicing appear in heterochromatin [33, 34]. Thus, at the blastoderm stage, heterochromatin rapidly acquires compaction and its characteristic structural and functional features. Interestingly, if at the blastoderm stage the embryo nuclei were transplanted into the cytoplasm of an unfertilized egg, the chromocenters rapidly disappeared and chromosome material was again transformed into a finely dispersed network [35]. Note that RUSSIAN JOURNAL OF GENETICS
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about one hour after heterochromatin becomes cytologically identifiable, the PEV spreading of its inactive state to euchromatic regions commences [36]. This stage (heterochromatin formation) coincides with the temperature-sensitive period for euchromatin compaction and genetic inactivation upon PEV [37, 38]. The extent of heterochromatin-mediated silencing attained in early embryogenesis may change during the subsequent development. According to [36], PEV is relaxed (i.e., genetic suppression reduces) in differentiating cells at the stage of completion of mitoses. Ontogenetic programming of the heterochromatin formation in chromosomes is confirmed in studies of other species. Karyological studies conducted on fishes, amphibians, and mammals convinced Prokofyeva-Belgovskaya [39] that chromosome morphology in the early embryogenesis is very different from that at the later stages: these chromosomes are less dense and not differentiated into eu- and heterochromatin. Prokofyeva-Belgovskaya referred to these chromosomes as juvenile ones. Telomeric heterochromatin. Ample cytological evidence indicates that heterochromatin is present in telomeric chromosome regions. Size of the heterochromatic blocks widely varies not only within species but also within populations (see [6, 40] for review). Telomeric heterochromatin, similarly to pericentric one, can be detected in Drosophila chromosomes beginning from early embryogenesis [33]. In all species examined, telomeric heterochromatin is represented by long tracts of DNA repeats (review in [41]). Telomeric heterochromatin produces a silencing effect on inserted transposons with reporter genes, which is analogous to PEV spreading. However, the telomeric position effect differs from PEV by specific impact of position effect modifiers [42]. Heterochromatization of the mammalian X chromosome. Only one of the X chromosomes of mammalian females (the euchromatic one) is transcriptionally active while the other becomes heterochromatic at the early embryonic stages, forming sex chromatin (Barr’s body) [43]. Most genes in this chromosome are transcriptionally inactive. The key regulatory element of dosage compensation in mammals, locus Xic (X inactivation center), was mapped using X-autosomal translocations. Analogously to PEV [44] inactivation spreading, the Xic locus translocated to autosomes suppresses adjacent genes, which gives grounds to consider these two phenomena similar. In mammals, the key regulatory gene for dosage compensation is Xist located in the inactivation center. The X chromosome carrying the Xist gene is suppressed. Xist transcription starts in the early development, and Xist RNA binds to the inactive X chromosome along its entire length (review in [7]). The material of the inactivated X chromosome exhibits many properties of heterochromatin [7, 19, 44–48]. 2003
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In extraembryonic tissues—trophoectoderm and primitive endoderm—the Xist expression is confined to the paternal X chromosome; consequently, this chromosome becomes heterochromatic [48]. Thus, X-chromosome silencing is strictly ontogenetically programmed. Paternal genome heterochromatization in coccides. In males of mealy bug species Pseudococcus citri and P. obscurus, the paternal genome becomes heterochromatic in the early development, later exists as an inactive element, and is finally destroyed prior to the formation of germ cells. Thus, only the maternal genome remains transcriptionally active and euchromatic, and only this genome is transferred to the offspring of either sex (sons and daughters). In daughters, the paternal genome remains euchromatic and in sons it becomes heterochromatic, and the scenario repeats [19]. The heterochromatic set of paternal chromosomes is readily identified in the interphase nuclei as an intensely stained chromocenter [49–52]. The heterochromatization of the paternal genome is often referred to as genetic imprinting. Thus, in all of the discussed cases cytologically identifiable heterochromatin exhibits similar structural (dense packing) and functional (splicing) properties. This suggests that heterochromatin as a cytological structure is the manifestation of splicing at the chromosomal level. The heterochromatin formation is strictly programmed in the development. The similarity between different heterochromatic regions is not confined to the above characteristics. In what follows, we consider other properties shared by heterochromatic regions. LATE REPLICATION, UNDERREPLICATION, AND ELIMINATION OF HETEROCHROMATIN IN SOMATIC CELLS It has long been known that different chromosome regions differ in the time of DNA replication [53]. For many animal, plant and yeast species, the completion of replication of heterochromatic regions was shown to be delayed as compared to the euchromatic regions (see [6, 9, 19, 22, 43, 45, 54] for review). Since this rule has practically no exceptions, Back [20] even stated that late replication is a single reliable diagnostic property of all types of heterochromatin. According to the widely spread view, the replication delay is explained by dense heterochromatin packing. The association between the structure and replication mode of the region is confirmed by the data on the time of appearance of late replication in ontogeny: it coincides with the time of heterochromatin “maturation.” For instance, in Drosophila heterochromatic regions become late replicating after a 14-day embryonic mitotic cycle, exactly at the stage when they undergo compaction [55, 56]. Euchromatic regions, which when intact are replicated without delay, change
replication mode upon their PEV compaction; i.e., they become the most late replicating in the genome [57]. The heterochromatic paternal genome of mealy bugs also replicates in the latter half of the S-stage [19, 58]. Sequence analysis of the “early” and “late” replication origins did not detect any clear differences between them. If a “late” origin, which is localized in telomeric heterochromatin, is transferred into an euchromatic region, it changes into an “early one” (see [47] for references). All this evidence confirms the conclusion that the mode of replication is determined by the heterochromatin structure rather than its nucleotide sequence. A very specific trait of heterochromatic regions is the underrepresentation of their DNA in somatic cells as compared to germline cells. It has been conclusively demonstrated that in Drosophila tissues with polytene chromosomes, DNA from pericentric heterochromatin and heterochromatic Y chromosomes is underreplicated. Heterochromatin constitutes about 30% of the metaphase chromosomes; in polytene chromosomes, most of it is not polytenized. Heitz and Painter first reported this phenomenon in 1933; since then, many data confirming the fact of heterochromatic DNA underreplication during polytenization has been obtained (reviews in [6, 8]). Not only pericentric but also telomeric regions are underreplicated (see [6] for references). Interestingly, euchromatic regions that underwent PEV compaction become not only late-replicating but also incompletely polytenized (review [6]). Heterochromatin underreplication is genetically controlled. The mutation SuUR (Suppressor of Underreplication) that suppresses underreplication of heterochromatin, including pericentric one, was found [59]. Homozygotes for this mutation have new banded regions in the chromosome arm bases as a result of the partial restoration of pericentric heterochromatin polytenization. Mutation suUR also supresses underreplication upon position effect (Belyaeva et al., unpublished data). The heterochromatin underreplication suppression in the SuUR mutant is probably caused by its earlier replication at the S stage [60]. Note that the underreplication-suppressing effect of the SuUR mutation does not involve all pericentric heterochromatin since most of it remains incompletely polytenized. Other examples of heterochromatin loss in somatic cells are known. They include different forms of chromatin diminution or elimination of total chromosomes and their fragments. Chromatin diminution is described mainly for Ascaris, Cyclops, and paramecia [61–63]. The most general characteristics of chromatin diminution are (1) the time at the very beginning of the embryonic development (second-third cleavage division) and (2) elimination of heterochromatic chromosome fragments [61, 64]. Finally, another interesting feature common for different heterochromatic genome parts is their localization on the inner side of the nuclear envelope. Using
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special optic technique, it was demonstrated that in Drosophila, about 30 polytene chromosome regions are more often located at the periphery of nuclei in cells of intestine and salivary and prothoracal glands. These regions include telomeric parts of chromosomes X, 2L, 3L, 3R and pericentric heterochromatin [65–67]. It was also found that the receptor B domain localized on the inner nuclear membrane binds to specific heterochromatic proteins HP1hsa and HP1hsy homologous to HP1 of Drosophila [68]. The chromo domain of the HP1 protein also participates in this interaction [69] (see below). In yeast, the nuclear envelope contains telomeric DNA: protein RAP1 binding to a telomeric repeat; telomeric proteins Ku70 and Ku80; structural suppressed chromatin proteins SIR3 and SIR4, and late replication origins [69a]. MOLECULAR MECHANISMS OF THE HETEROCHROMATIC DOMAIN FORMATION AND SPLICING As early as in 1974, Zuckerkandl (see [6]) suggested that the global heterochromatin properties are caused by specific properties of its protein components determining its compact state rather than by specificity of its DNA. The development of this idea was largely related to the detection and analysis of genetic modifiers of position effect, i.e., genes whose mutations suppress (Su(var)) or enhance (E(var)) the inactivation of euchromatic genes transferred to heterochromatin. For this purpose, the easily tested reduction or enhancement of mosaic color in Drosophila eyes connected to the white gene position effect in a rearranged chromosome is typically used. The reduction or enhancement of PEV reflects spreading of inactive heterochromatic state in the transferred euchromatin; hence, PEV modifiers affect silencing, i.e., the organization of heterochromatin itself. Comparatively easy isolation of PEVmodifying mutations permitted to identify several tens of genes affecting heterochromatin organization ([70–72; see 4, 6, 12] for review). A high number of PEV modifiers and the dose dependence of their effect laid the grounds for a hypothesis on specific protein complexes that bind to heterochromatic DNA causing its compaction and silencing. It was assumed that the assembly of the protein complexes in heterochromatin starts in “inactivation centers” and then spreads over great distances thus ensuring spreading of the heterochromatin suppression effect upon PEV [73]. This hypothesis has been brilliantly confirmed by the studies of molecular mechanisms of splicing that recently has been rapidly progressing. These studies involve different models (yeast, Drosophila, mammals, etc.) and demonstrated integrity and conservatism of the processes underlying genetic silencing in all organisms, from yeast to human. Below, we consider main advances in the field of studying the molecular and structural organization of heterochromatin. RUSSIAN JOURNAL OF GENETICS
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The main structural unit of chromatin is the nucleosome, whose 146-bp DNA winds around the core octamer of highly conserved histone proteins (two copies of each of H2A, H2B, H3, and H4). Nucleosomes are linked together by linker DNA. They in some way participate in any activity of nuclear DNA, including transcription, replication, and repair. A comparison of the nucleosome organization of euand heterochromatic regions showed that heterochromatic nucleosomes are regularly spaced at a considerable distance. The nucleosome organization in euchromatin is more irregular. These differences may be related to a more “closed” conformation of heterochromatin [10, 74, 75]. Heterochromatin is considerably more resistant to the effect of both nonspecific (DNase I and micrococcal nuclease) and specific (restriction endonucleases) nucleases; it has significantly lower number of nucleosome-free, hypersensitive to nucleases sites characteristic for active genes [10, 76, 77]. These data suggest that silencing involves alterations in euchromatin packing not only at the level of the higher-order structure but also at the level of the nucleosome organization. The results demonstrating specificity of posttranslational modifications of heterochromatic histones are even more significant for understanding the mechanisms of silencing. The N- and C-ends (tails) of core histones can undergo various modifications: hypoacetylation, methylation, phosphorylation, ubiquitinization, and ATP-ribosylation. These modifications occur in different amino acids (Fig. 2). The example of modifications of a histone molecule is called the histone code. The histone code hypothesis implies that these modifications are prerequisites for the subsequent change in the chromatin structure and preserve the mode of gene expression in DNA replication and cell division. In addition to histone modification, an important role in forming silent domains is played by cytosine methylation [8, 10, 78–86]. Hyperacetylated histones (which we do not consider here) are characteristic for euchromatic domains whereas heterochromatic histones are hypoacetylated. This explains the functional role of histone acetyltransferases (HAT) and deacetylases (HDAC) in the regulation of gene expression. This enzyme activity is present in many transcription regulation factors. In most of the examined species, the major sites of histone H3 acetylation are lysine molecules at different positions (Fig. 2). Methylation of the histone tails of the nucleosome core is produced by methyltransferases specific for lysine (K-HMT) or arginine (R-HMT). Transmethylase methylates lysine of histone H3 at position 79. Lysine 79 is located at the surface of the core octamer where methylation has a potential effect on the interaction with other proteins. Methylation of lysine 9 in histone H3 is among the most significant characteristics of the heterochromatic state in Drosophila [87–90] and silencing of the mating type locus in yeast [27]. The 2003
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Fig. 3. The structure of protein HP1 in Drosophila (after [10]; see text). Reprinted from Richards, E.J. and Elgin, S.C.R., Epigenetic Codes for Heterochromatin Formation and Silencing: Rounding up the Usual Suspects, Cell (Cambridge, Mass.), vol. 108, pp. 489– 500, copyright 2002, with permission from Elsevier Science.
modified histone mH3K9 has been found in this locus (an analog of pericentric heterochromatin) but not in the euchromatic region flanking it. The major role of the H3 histone methylation in the formation and spreading of silencing is evidenced by the fact that the deletion of specific repeated sequences normally located at the edges of the mating type locus, leads to the extension of silencing to the neighboring euchromatic regions [27]. This situation is analogous to the PEV silencing spreading in Drosophila (see above). Histone mH3K9 antibodies are localized in polytene
Drosophila chromosomes in the pericentric (chromocenter) and telomeric regions as well as in several euchromatic arm sites and in chromosome 4 [52, 88]. In the murine metaphase chromosomes mH3K9 is detected in pericentric heterochromatin and G-bands and co-localizes with the late replication regions [52]. In addition to the modified histones, heterochromatin harbors specific nonhistone genes, many of which are products of PEV modifier genes. At least four proteins, HP1, SU(VAR)3-7, SU(VAR)3-9, and SUUR, are always found in pericentric heterochromatin [88, 91–95].
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Data on other heterochromatic proteins are presented in the review [93]. Some of these proteins were examined in detail and their role in forming heterochromatic domains was shown. For instance, it was found that the Drosophila SU(VAR)3-9 protein (the product of gene Su(var)3-9 whose mutations suppress PEV) is methyltransferase methylating lysine 9 of histone H3. The antibodies to SU(VAR)3-9 are located in pericentric heterochromatin and on chromosome 4 [92]. The protein is highly conserved; Su(var)3-9 homologs were detected in yeast (Clr4), mouse (Suv39h1), and human (SuV39H1). All these genes encode methyltransferases methylating lysine 9 in histone H3 and are localized in pericentric heterochromatin [92, 96–98]. The methyltransferases contain chromo- and SET-domains [13, 99–101]. Mutations in the SET-domain consisting of 130–160 amino acids disturb the methylating activity of the protein. HP1 (Heterochromatin Protein) is a specific heterochromatic protein studied in most detail [10, 93]. It is encoded by a PEV-modifying gene Su(var)2-5. HP1 appears in the nuclei of early Drosophila embryos at the stage when heterochromatin begins to be detected in chromosomes. In polytene chromosomes it is localized in pericentric heterochromatin, telomeres, chromosome 4, and some sites of euchromatic arms. The HP1 protein was also found in the euchromatic regions turned into heterochromatic via PEV [33, 102–104]. The HP1 protein contains two conserved regions at the N- and C-ends: chromo domain and chromoshade domain (Fig. 3). Chromo domain mutations lead to the loss of protein activity. The protein is highly conserved, its homologs were found in various eukaryotic organisms, from yeast to human (see [93] for review). HP1 interacts with many other chromosomal proteins; mutations in the chromoshade region deprive it of this ability. The N-terminal chromo domain is required to bind HP1 to the methylated H3 histone. It is assumed that the HP1 binding to the modified H3 histone is required to stabilize the suppressed state of heterochromatin [10]. The complex functioning of proteins HP1 and SuV39H1 is evidenced by their coimmunoprecipitation [92, 105] and behavior in mutants. For instance, the Su(var)3-9 mutation in Drosophila leads to a HP1 disappearance from the chromocenter. Conversely, the Su(var)2-5 mutation (gene Su(var)2-5 encodes HP1) disrupts the normal distribution of the SU(VAR)3-9 protein, which is redistributed from the chromocenter to numerous regions of euchromatic chromosome arms [105]. Interestingly, another PEV-modifying gene, pitkin (ptn), has a similar effects. Heterozygotes for the ptnD mutation also exhibit SU(VAR)3-9 redistribution from the pericentric heterochromatin to euchromatic regions of polytene chromosomes [106]. Apparently, the ptn gene product also participates in the formation of protein complexes responsible for silencing. Another component of these complexes is probably protein SU(VAR)3-7, which coimmunoprecipitates with HP1 in embryonic Drosophila extracts and is localized in the RUSSIAN JOURNAL OF GENETICS
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Heterochromatin formation Fig. 4. Stages of heterochromatin assembly in locus MAT of yeast S. pombe (after [10]). CD, chromo domain; CSD chromoshade domain (see text). Reprinted from Richards, E.J. and Elgin, S.C.R., Epigenetic Codes for Heterochromatin Formation and Silencing: Rounding up the Usual Suspects, Cell (Cambridge, Mass.), vol. 108, pp. 489–500, copyright 2002, with permission from Elsevier Science.
same heterochromatic regions as HP1 [91, 107]. SU(VAR)3-7 contains seven zinc finger motifs, which implies its binding to DNA. Other heterochromatin-associated proteins are known that potentially can take part in heterochromatin assembly, for example, the product of the PEV-suppressing gene modulo [108]. The SUUR protein is also interesting in this respect. This protein is located in late-replicating regions of polytene chromosomes, including chromocenter and PEV-heterochromatized regions ([94]; E.S. Belyaeva et al., unpublished data). SUUR has homology with the N-terminal part of the ATP-helicase domain from the SNF2/SWI2 protein family possessing remodeling properties [94]. It can be assumed that the SUUR protein, which controls DNA underreplication in polytene Drosophila chromosomes, interacts with the silencing complex as a chromat5inremodelling factor. Of course, the current picture of molecular events resulting in silencing is far from complete but its general features are becoming clear. The silencing mechanisms are studies in most detail for telomeric regions and MAT (mating type) locus in yeast [10, 22, 41, 78]. The sequence of events leading to silencing is show on the diagram of heterochromatin formation in locus MAT (Fig. 4). Histone H3 deacetylation by specific 2003
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Fig. 5. Temporal characteristics of different events taking place in cultured stem embryonic cells upon X-chromosome inactivation in female mammals (after [48]). Reprinted from Brockdorff, N., X-Chromosome Inactivation: Closing in on Proteins that Bind Xist RNA, Trends in Genetics, vol. 18, pp. 352–358, copyright 2002, with permission from Elsevier Science.
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Fig. 6. Binding of DNA methyl transferase (Dnmts) to a specific chromosome region. X, DNA-binding transcription factor (or corepressor), which can attract Dnmt. Protein HP1 binding to methylated histones also may attract Dnmt although this has not been demonstrated yet (after [116]). Reprinted from Burgers, W.A., Fuks, F., and Kouzarides, T., DNA Methyltransferases Get Connected to Chromatin, Trends in Genetics, vol. 18, pp. 275–277, copyright 2002, with permission from Elsevier Science.
deacetylases Clr6 and (or) Clr3 creates conditions for methylation of lysine 9 (K9) by the Clr3/Rik1 complex (methyltransferase and DNA-binding protein required for methylation acceleration). Methylation of histone H3 results in its binding with the Swi6 protein homologous to HP1 followed by the assembly of the stable heterochromatin structure inaccessible for transcription factors (see review [10] for details). In general, these three main stages of silencing are shared by various organisms and heterochromatin types studies although they possess some specific features. Silencing of the mammalian X chromosome is accompanied by hypoacetylation of histones H2A, H3, and H4 [48, 109, 110] and methylation of H3K9 [10, 48, 52, 84, 110–112]. The consecutive stages of X-chromosome inactivation in female mammals are shown in Fig. 5. However, no HP1 homologs were found in the inactive X chromosomes, and silencing of the imprinted paternal X chromosome in extraembryonic tissues involve
the product of locus eed, a homolog of the extra sex comb gene of Drosophila belonging to the Polycomb gene group [113]. The product of this gene is thought to be required for attracting histone deacetylases [114]. The signs of X-chromosome inactivation in mammalian embryonic cells appear gradually in a narrow time interval of early embryonic development [48, 115]. Silencing of the X chromosome and other types of heterochromatin in plants and animals involve methylation of DNA cytosine [10, 84, 116], which is, however, not characteristic of insects, e.g., Drosophila. Postreplicative cytosine methylation is effected by various groups of DNA methyltransferases (Dnmt). Three active DNA methyltransferases (Dnmt1, Dnmt3a, and Dnmt3b) are known in mammals. It was shown that the N-terminal noncatalytic Dnmt1 domain binds to deacetylase and can suppress gene transcription via deacetylase activity while the enzyme methylates the DNA cytosine (Fig. 6).
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Characteristics of phenomenon Packing of chromosome material Chromatin structure
Pericentric heterochromatin
Dense, heteropicnic
Dense, heteropicnic
ND
Lower accessibility to nucleases
Regular arrangement ND of nucleosomes, lower sensitivity to nucleases
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Low density Heterochromatic genes are induced Termination of DNA rep- Late replication lication Chromatin diminution or DNA underreplication underreplication in somat- in polytene chromosomes; ic cells chromatin diminution Underreplication suppres+ sion by mutation SuUR Localization in nucleus Mainly on nuclear envelope PEV induction + DNA sequences Satellites, mobile elements Histone modifications: + histone deacetylation and H3K9 methylation Nonhistone proteins: HP1 + + + – +
– –
Dense, heteropicnic
High density Silencing state
High density Silencing state
Late replication
Late replication
Late replication
DNA underreplication in polytene chromosomes ND
DNA underreplication ND in polytene chromosomes + ND
Mainly on nuclear envelope + Specific repeats
ND
+
– Mainly unique
+ – – – +
+ ND ND ND
In the whole nucleus ND – Mainly unique Mainly unique
+
+
+ ND
ND
+
ND +
–
+
+
Different
Irregular arrangement of nucleosomes; sensitivity to nucleases High density High density Silencing state Euchromatic genes are induced Late replica- Throughout Stion phase ND ND
ND
Mainly on nuclear envelope + Mainly unique
ND
Dense, heteropicnic ND
Euchromatin
ND ND ND ND
–
Dispersed localization – – + –
141
Note: ND, no data.
Heterochromatization Inactivated Inactivated X chromoof euchromatin at posimale genome some in female mammals tion effect variegation in coccids
Dense, heteropicnic
Presence of genes State of gene activity
SU(VAR)3-9 SU(VAR)3-7 PcG SuUR
Telomeric heterochromatin
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Comparison of the properties of pericentric heterochromatin, euchromatin at position effect variegation, inactivation of the X chromosome in female mammals, and the genome of male coccids (see text for references and details)
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Other details of the DNA methyltransferase action producing silencing are poorly understood. It is known that the state of DNA methylation can be dynamic. The best example of that is the disappearance and reappearance of the DNA methylation regime in early development of mammals (see [10, 116] for references). Some other organism-specific features of silencing have been established. For instance, system mH3K9/HP1 critical for the pericentric heterochromatin formation in Drosophila, is less significant for telomeric silencing in yeast, which suggests the existence of other, additional mechanisms underlying inactivation of these regions [117]. CONCLUSION The evidence on mechanisms of genetic silencing that we attempted to overview in this paper are far from sufficient to construct an integral picture of this complicated phenomenon. However, even now it is becoming apparent that these mechanisms are essentially similar in all eukaryotes (table) and, which is of particular importance, participate in assembly of different types of heterochromatic regions. Silencing is accompanied by alteration of the chromatin structure beginning from the nucleosome level, making it compact and inaccessible for transcription factors. At the chromosome level, silencing of extensive chromosome regions is manifested in cytologically identifiable heterochromatin. There are indications that similar heterochromatin transformations happen also at stable, inherited in cell generations inactivation of particular genes whose suppression is required for normal development. Thus, silencing is a basic epigenetic mechanism ensuring differential gene expression in individual development of all eukaryotic organisms, from yeast to human. We should like to emphasize that silencing is not a permanent state of heterochromatic regions. In all of the cases considered above, silencing is strictly determined and begins at a particular stage of early embryonic development. Transcription in the chromosomes of the developing embryo starts exactly at this stage. The assembly of heterochromatic domains thus safely protects organisms against the expression of genetic material that is not needed in the somatic cells. “Whereof one cannot speak thereof one must be silent” [15]. The primary DNA structure does not restrict targets of silencing. The latter involves both repetitive (pericentric and telomeric heterochromatin) and unique sequences (PEV, X chromosome of female mammals, paternal coccid genome), although repeats are likely to promote heterochromatin formation. The functions of highly repetitive heterochromatin remain enigmatic. This heterochromatin may be underreplicated or removed from somatic cells (diminution), which testifies to the absence of any important cell function. Nevertheless, all heterochromatin is pre-
served in the germline genome and is thus transferred in generations as a species-specific character. The commonness of molecular mechanisms of genetic suppression underlies similarity of different heterochromatic regions, their compactness, staining, replication time, etc. The phenomenon of position effect variegation that has long remained mysterious is also becoming clearer: PEV-producing rearrangements disrupt the barrier that normally divides eu- and heterochromatin, and the protein complexes ensuring silencing in pericentric heterochromatin spread to the transferred euchromatin. Note that the modern views on heterochromatin discussed here were in essence advanced by Prokof’evaBel’govskaya as early as in the 1930s–1940s; this author regarded heterochromatin as a “state” rather than a special “substance” ([118]; see also [7, 13]). Finally, some words on terminology should be added. In view of the common mechanism of silencing for various heterochromatic elements of the genome, similarity of their characteristics and time of appearance in the development, classification of heterochromatin into constitutive and facultative that was proposed by Brown [19] and has been widely used in current literature seems incorrect since it is not based on principal differences between different types of heterochromatic regions. ACKNOWLEDGMENTS We are grateful to S. Henikoff, T.B. Nesterova, and S.M. Zakiyan for assisting with literature; N. Brockdorff, S. Elgin, E. Richards, P. Paro, T. Kouzarides, and M. Hampsey, for kindly providing electron versions of the figures and permitting their use; and D.E. Koryakov and E.A. Dolbak, for help in the preparation of the manuscript. This study was supported by the Russian State Program “Frontiers in Genetics” (grant no. 2-02PNG-2002), Russian Foundation for Basic Research (grant nos. 00-1597984 and 02-04-48222), INTAS (grant no. 99-1088), and Ministry of Higher Education of Russian Federation (grant no. PD02-1.4-74). REFERENCES 1. Heitz, E., Das Heterochromatin der Moose, Jb. Wiss. Bot., 1928, vol. 69, pp. 762–818. 2. Heitz, E., Der Bau der somatischen Kerne von Drosophila melanogaster, Z. Indukt. Abstammungs-Vererbungslehre, 1930, vol. 54, pp. 248–249. 3. Muller, H., Types of Visible Variations Induced by X-rays in Drosophila melanogaster, J. Genet., 1930, vol. 22, pp. 299–334. 4. Wallrath, L.L., Unfolding the Mysteries of Heterochromatin, Curr. Opin. Genet. Dev., 1998, vol. 8, pp. 147– 153.
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2003