BIOLOGICAL OVERVIEW

Distinct modifications of histone amino termini, such as acetylation, phosphorylation and methylation, have been proposed to underlie a chromatin-based regulatory mechanism that modulates the accessibility of genetic information. Histone lysine methylation occurs on lysines 4, 9, 27, 36, and 79 of Histone H3 and on lysine 20 of Histone H4. Biochemical and genetic studies indicate that methylation of different lysine residues, with the exception of H3-K79, is catalyzed by different SET domain-containing proteins. Histone H3 methylated at lysine 9 (H3-mLys9) is characteristic of the heterochromatic (genetically silent) state. Immunofluorescent staining of Drosophila polytene chromosomes shows that the bulk of the H3-mLys9 is present in the pericentric heterochromatin and in a banded pattern on the fourth chromosome, known sites of repetitive DNA. Similarly, chromatin immunoprecipitation experiments demonstrate that H3-mLys9 is a prominent component of the silent mating type locus in fission yeast (Schizosaccharomyces pombe), while essentially absent from flanking regions containing inducible genes. Methylation of histone H3-Lys9 has also been associated with the silencing of euchromatic genes (Richards, 2002 and references therein).

A key role for gene silencing and specification of heterochromatin is shown by the demonstration that mammalian homologs of Drosophila Su(var)3-9, including human SUV39H1 and murine Suv39h1, encode enzymes that specifically methylate histone H3 on lysine 9. Su(var)3-9 was originally identified as a suppressor of PEV in Drosophila, indicating that the wild-type gene product is involved in heterochromatin formation. A homolog in S. pombe, Clr4, is also a specific histone H3-Lys9 methyltransferase, suggesting that this activity is widely distributed and well conserved. clr4 mutants exhibit reduced heterochromatin formation at centromeres, with elevated mitotic chromosome loss and reduced silencing within both pericentromeric heterochromatin and the silent mating type locus. Similarly, mammalian Su(var)3-9-like proteins have been implicated in both centromere activity and gene silencing. Disruption of the murine Suv39h1 and Suv39h2 paralogs causes genome instability, chromosome missegregation, and male meiotic defects (Richards, 2002 and references therein).

Drosophila Polycomb Group (PcG) complexes are responsible for the maintenance of the repressed state of genes subject to their control. The best-known PcG targets are the homeotic genes, which are activated in the early embryo by the products of segmentation genes. At this stage, transient, localized activators and repressors determine the segmental domains of expression of each homeotic gene but, after gastrulation, epigenetic (genetically based non-heritable) mechanisms take over to maintain the segmental pattern of expression for the rest of development. These mechanisms are mediated by the Polycomb Response Elements (PREs), regulatory regions of several hundred base pairs, where two kinds of chromatin complexes are assembled. One kind, the PcG complexes, is repressive and can maintain a silent state. The other kind involves the Trithorax protein (Trx) and mediates the persistence of the active state. Which of the two predominates depends on the state of activity of the target promoter at the blastoderm stage of development. If the genes had been repressed by early regulators, the PcG silencing mechanisms maintain the repressed state throughout the rest of development. If the gene was active in the early embryo, the Trx function stimulates its expression and prevents later silencing by the PcG complexes. The gene remains then unrepressed and potentially active for the rest of development. Thus, early transcriptional activity of a target gene sets a mark that maintains transcriptional competence and prevents the establishment of PcG silencing, while early repression results in an antagonistic mark that ensures the maintenance of the silenced state in subsequent cell cycles (Czermin, 2002 and references therein).

Formally, therefore, the PRE mediates both the memory of the silent state and the memory of the derepressed state. The repressive memory is illustrated by the fact that, although PcG proteins are nearly ubiquitous, at every cell division they restore the repressed chromatin state only in cells in which their target genes had been previously repressed. The memory of the derepressed state is shown by the fact that if the target gene is active in the early embryo, or if derepression is forced by massive doses of activator, the derepressed state is inherited by the progeny cells. This memory is affected by trx mutations. In the absence of trx function, cells in which a PRE-containing construct had been activated in the early embryo may lose the derepressed state and become silenced again (Czermin, 2002 and references therein).

In the preblastoderm embryo, Polycomb complexes are assembled at the PREs, which contain consensus sequences for DNA binding proteins such as GAGA factor and Pleiohomeotic (Pho), the fly homolog of the mammalian YY1 factor. These, together with other, unidentified DNA binding proteins, recruit cooperatively a PcG complex that includes Pc, Ph, and GAGA factor but also Esc, E(z), Pho, and Rpd3. The E(z)/Esc/Pho complex dissociates from the Pc-containing complex after the blastoderm stage, and Esc ceases to be produced by the end of embryogenesis. However, the E(z) protein continues to be needed, at least intermittently, to maintain the silent state and is most likely recruited to the PRE by the Pho DNA binding protein. Experiments with Pc targeted to a reporter gene by the LexA DNA binding domain show that, while it can recruit the Esc/E(z) component to establish silencing in the early embryo, it can no longer recruit E(z) at later stages, when the early complex has dissociated. LexA-Pc repression continues in the embryo but the memory of the repressed state is then lost and the reporter gene becomes derepressed during larval stages. These results suggest that E(z) might mediate the creation of a chromatin mark necessary for repression and responsible for maintaining the memory of the silent state (Czermin, 2002 and references therein).

Trx and E(z) are therefore good candidates for the functions required for the positive and negative memories, respectively. Structurally, these two proteins share with Su(var)3-9 the SET domain, named after the three founding members Su(var)3-9, E(z), and Trx. Advances in the past years have shown that the SET domain in many proteins is responsible for a histone H3 methyltransferase (MTase) activity. With one exception, all reported histone MTases that methylate lysine residues contain a SET domain, which harbors the amino acids important for MTase function. Su(var)3-9 and its homologs, in particular, are necessary for the formation of heterochromatic complexes in mammals, flies, and fission yeast. Their activities methylate lysine 9 of histone H3, which becomes a binding site for the chromodomain of heterochromatin proteins such as Hp1 (Czermin, 2002 and references therein).

It was reasoned that Trithorax and Enhancer of zeste might reside in complexes that possess MTase activities. Pc contains a chromodomain whose structure and essential residues are homologous to those found in Hp1 and related methyl lysine binding proteins and might therefore recognize a nucleosomal methylation mark. Both Trx and E(z) complexes have now been shown to contain an H3 MTase activity. To study the Esc/E(z) complex, Drosophila nuclear extract was fractionated and it was asked if a MTase activity copurifies with E(z) and Esc. A complex containing E(z) and Esc trimethylates lysine 9 and methylates lysine 27 of histone H3 and the trimethylated lysine 9 mark is closely correlated with PcG binding sites on polytene chromosomes. The conjecture that the Esc/E(z) complex contains a histone MTase activity has been confirmed by the finding that the complex immunoprecipitated by anti-Esc or purified biochemically methylates in vitro histone H3 whether assembled in a nucleosome, as a free histone, or in the form of oligopeptides. The purified complex contains several components as predicted from previous studies: in addition to E(z), SU(Z)12, Esc, p55, and Rpd3 are found. An additional component of approximately 168 kDa remains to be identified (Czermin, 2002).

The activity of the Esc/E(z) complex leads to trimethylation of lysine 9 and probably also of lysine 27 of H3. In vivo, an antibody directed against dimethylated H3 lysine (9me2K9 antibody) does not detectably stain chromosomal PcG sites while a me3K9 antibody directed against trimethylated H3 lysine decorates all chromosomal PcG sites. Whether three methyl groups are added processively or by independent events and whether lysine 9 and lysine 27 are targeted simultaneously remains to be elucidated. The partial ability of the complex to methylate the peptide acetylated at lysine 9 is accounted for by the presence of the Rpd3 deacetylase (Czermin, 2002).

The Su(var)3-9 product has been reported to dimethylate H3 lysine 9, constituting a mark for heterochromatic complexes (Rea, 2000; Lachner, 2001). However, antibody staining of polytene chromosomes detects abundant trimethyl lysine 9 in heterochromatin as well as most of chromosome 4, sites that do not contain PcG complexes. At least some of the heterochromatic me3K9 is lost in Su(var)3-9 mutants. A participation of E(z) in heterochromatic H3 methylation cannot be excluded. This function would be consistent with the report that E(z) mutations are also suppressors of heterochromatic position-effect variegation. However, a more distinct difference between the Su(var)3-9 mark and the E(z) mark is the methylation of H3 K27. In vitro, Su(var)3-9 does not methylate the K27 peptide. It will be important to test whether K27 methylation is present in heterochromatin, but it is likely that K27 methylation differentiates heterochromatic from PcG sites (Czermin, 2002).

Suvar3-9 methylation of lysine 9 of histone H3 is thought to stabilize or even target Hp1-containing heterochromatic complexes through binding of the Hp1 chromodomain to the methylated lysine 9 (Bannister, 2001; Lachner, 2001). The structure of the Hp1 chromodomain bound to histone H3 di- or tri-methylated at lysine 9 shows that either peptide fits in a groove and lodges the methyllysine in a hydrophobic pocket (Nielsen, 2002; Jacobs, 2002). This was confirmed by peptide binding experiments and makes it unlikely that H3 K9 methylation would be sufficient to discriminate between the heterochromatic methylation imprint and the PcG methylation imprint. The parallelism suggests that the chromodomain of Pc would recognize trimethyl lysine 9 H3. Pc has been shown to bind to histone H3 and to nucleosomes in vitro; however, the domain involved does not appear to be the chromodomain but the C-terminal region (Breiling, 1999). Binding experiments detected little increased affinity of Pc for the trimethyl K9 peptide compared to the unmethylated peptide. Instead, methyl K27 appears to make the major contribution to Pc affinity for methylated H3. The amino acid context of K27 (KAARKS) resembles that of K9 (QTARKS) but Hp1 binds weakly to a methylated K27 peptide (Nielsen, 2002; Jacobs, 2002). It remains to be seen which domain of Pc interacts with methylated H3 and whether Hp1 can bind to meK9 meK27 H3. Nevertheless, the presence of me3K9 at PcG sites, as well as in heterochromatin and chromosome 4, suggests that the meK27 or other factors must contribute to discriminate between heterochromatic and PcG sites (Czermin, 2002).

Specific recognition of the methylated histone H3 by Polycomb complexes might be facilitated by other PcG components or by other modifications of the histones. E(z)-dependent methylation might contribute to the stability of the PcG complex, particularly in the early stages of assembly at the PRE, for example, by permitting complex formation to spread to neighboring sequences for a distance of 2- 3 kb. However, the fact that in the E(z)^S2 mutant chromosomes the trimethylation mark is lost well before the binding of PcG proteins indicates that methylation is not essential for the binding of the complex. Alternatively, the trimethyl mark might signify the difference between the mere recruitment of a PcG complex and its repressive function. Chromatin immunoprecipitation shows that PcG complexes are present at some PREs whether or not the corresponding gene is repressed, implying that, while recruitment of the complexes may be constitutive, the decision to repress or not depends on other features transmitted epigenetically. The methylation might then constitute the epigenetic mark triggering the silent state. It is interesting to note, therefore, that two polytenic sites that are strongly stained with anti-Psc antibody but very weakly with anti-me3K9 antibody are 2D and 49F, respectively, the sites of the PcG genes ph and Psc. These PcG genes are downregulated but not silenced by PcG mechanisms. If trimethylation of H3 lysine 9 signals strong silencing, these sites might be expected to be occupied by PcG complexes but only partly repressed (Czermin, 2002).

A role of H3 methylation in the assembly of stable PcG complexes is suggested by another observation. When the chromodomain in Hp1 is substituted by the chromodomain of Pc, the chimeric Hp1 is recruited to PcG binding sites on polytene chromosomes. This implies either that the chromodomain is sufficient to recognize and bind to the meK9 meK27 H3 or that the Pc chromodomain specifies critical interactions with other PcG components that are recruited to PcG sites. That the latter interpretation is correct is shown by the fact that the chimeric Hp1 also recruits PcG proteins to heterochromatin, where they are not normally found, and that this recruitment is dependent on E(z) function. Strong staining is observed with the anti-me3K9 antibody in the chromocenter and chromosome 4, implying that me3K9 H3 is widespread in heterochromatin. If this is, in fact, due to E(z) activity, it would explain the intervention of E(z) in the heterochromatic recruitment of PcG proteins mediated by the chimeric Hp1 (Czermin, 2002).

It would be important, therefore, to determine whether E(z) contributes to heterochromatic me3K9. Anti-E(z) antibody does not stain the chromocenter of salivary polytenic chromosomes. In E(z)^S2 mutant larvae raised at nonpermissive temperature, me3K9 staining is still seen in heterochromatin although with very variable intensity. One possible explanation for these two observations is that since heterochromatin is very little replicated in polytene chromosomes, the methylated H3 produced before the temperature shift might perdure a long time at nonpermissive temperature (Czermin, 2002).

It is interesting to note that anti-me3K9 staining at telomeres is not affected in Su(var)3-9 null polytene chromosomes but is lost in the absence of E(z) function. Telomeres, particularly those of chromosome 2R and 2L, stain prominently with antibodies against PcG proteins but also against Hp1. A critical role has been attributed to telomeric Hp1 in 'capping' chromosome termini and preventing telomeric fusions, failure to segregate chromosomes, and chromosome breakage. The fact that Drosophila Su(var)3-9 null mutants are perfectly viable implies that this role of Hp1 is not dependent on Su(var)3-9. In contrast, embryos lacking both maternal and zygotic E(z) function are reported to have mitotic phenotypes similar to those attributed to Hp1 mutants. It is likely, therefore, that the recruitment of Hp1 to telomeric sites depends at least in part on E(z) and not on Su(var)3-9. Unfortunately, the evidence obtained with the E(z)^S2 mutant is inconclusive (Czermin, 2002).

It is concluded that a combination of histone methylation marks could be a major factor in the establishment of stable patterns of homeotic gene expression and constitutes the molecular basis of a cellular memory system. The analysis of the existing methylation patterns in cells expressing different homeotic genes by chromatin immunoprecipitation will give further insight into how the MTases such as E(z) are targeted to different sites and how they are regulated at different developmental stages (Czermin, 2002).

GENE STRUCTURE

4.8kb and 5.0kb repeats containing the histone genes His1, His2A, His2B, His3 and His4 were present in all of the more than 20 D. melanogaster strains studied. The strains differ in the relative amounts of the two repeat types, with the 5.0kb repeat always present in equal or greater amounts than the 4.8kb repeat. The strains also differ in a number of far less abundant fragments containing histone gene sequences. The expression of HIS-C genes, including His3, during oogenesis has been studied, and compared to periods of DNA synthesis and actin expression during this developmental stage. The D. virilis core histone genes (Dvir\His2B, Dvir\His3, Dvir\His4 and Dvir\His2A), are arranged in the same order and orientation as the D. melanogaster core histone genes (His2B, His3, His4 and His2A). However, the His1 gene that is located between His2B and His3 in D. melanogaster is not found between Dvir\His2B and Dvir\His3 in D. virilis. The genomic organization of the histone genes in D. hydei closely resembles that of D. melanogaster. The position of the homologous histone gene repeats within the nuclei of early embryo cells has been investigated. The two homologous histone gene clusters are distinct and separate through all stages of the cell cycle up to nuclear cycle 13. During interphase of cycle 14, the two clusters colocalize with high frequency, and move from near the midline of the nucleus towards the apical side. The codon bias of the histone genes from D. melanogaster and D. hydei illustrates that the generalization -- that abundantly expressed genes have a high codon bias and low rates of silent substitution -- does not hold for the histone genes. DNA replication of the 5kb histone gene repeating unit in tissue culture cells (Drosophila Kc cells) initiates at multiple sites located within the repeating unit. Several replication pause sites are located at 5' upstream regions of some histone genes. The TFIID complex interacts with the promoter of His3 making contacts at the TATA element, initiator, +18 and +28 regions. Distinct specific subsets of lysines are utilized during deposition-related His4 diacetylation (FlyBase record for His3 and references therein).

PROTEIN STRUCTURE

Amino Acids - 135

For information on H3 structure see Structures of Histone Proteins and Proteins Containing the Histone Fold Motif and associated links.

date revised: 18 November 2002

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