Suppressor of variegation 3-9
Mutations in the gene for Su(var)3-9 are dominant suppressors of position-effect variegation (PEV). Su(var)3-9 is shown to be a chromatin-associated protein and the large multicopy histone gene cluster (HIS-C) is identified as one of its target loci. The organization of nucleosomes over the entire HIS-C region is altered in Su(var)3-9 mutants and there is a concomitant increase in expression of the histone genes. Su(var)3-9 is a histone H3 methyltransferase and, using chromatin immunoprecipitation, it has been shown that Su(var)3-9 is present at the HIS-C locus and that histone H3 at the HIS-C locus is methylated. It is proposed that Su(var)3-9 is involved in packaging HIS-C into a distinct chromatin domain that has some of the characteristics of ß-heterochromatin. It is suggested that methylation of histone H3 is important for the chromatin structure at HIS-C. The chromosomal deficiency for the HIS-C is also a suppressor of PEV. In contrast to what might be expected, it is shown that hemizygosity for the HIS-C locus leads to a substantial increase in the histone transcripts (Ner, 2002).
An antibody was raised against an Escherichia coli expressed polypeptide representing one-third of Su(var)3-9 that includes the chromodomain region. The antibody appeared to detect Su(var)3-9 localized to the 39D-E region as well as to many other sites in the euchromatin. The immunolocalization of Su(var)3-9 to the 39D-E region is especially strong. Since this is the site of the large multicopy HIS-C, it raised the possibility that Su(var)3-9 is associated with this cluster of tandemly reiterated genes. HIS-C includes ~0.5 Mb of DNA and contains 110 copies of the five histone genes, and it occupies bands 39D2-3-39E1-2. Since the HIS-C genomic region and its chromatin structure are well defined, and its product is very abundant as well as essential, this locus provides an almost ideal site for examining the effect, if any, of Su(var)3-9 mutations on chromatin structure and gene expression (Ner, 2002).
The data show that mutations in a Su(var)3-9 alter chromatin structure and concomitantly alter gene expression. This is the first demonstration that mutations in a Su(var) gene actually alter chromatin structure and that this alteration in structure is associated with an alteration in gene expression. Even though the Drosophila Su(var)3-9 protein is 223 amino acids larger than its mammalian counterpart, this study shows that Drosophila Su(var)3-9 also has a histone H3 methyltransferase. Finally, the ChIP data indicate that a significant proportion of the H3 histones associated with the HIS-C locus are methylated and that the Su(var)3-9 protein associates with the HIS-C locus. Collectively, these data suggest that Su(var)3-9 is a trans-regulator of histone gene expression and that it regulates histone gene packaging and expression by forming part of the HIS-C chromatin rather than by transiently associating with the region (Ner, 2002).
These data highlight an intriguing discrepancy in the localization of Su(var)3-9 protein. Su(var)3-9 has been shown to localize to many euchromatic sites and to the chromocenter of polytene chromosomes. In addition, the pattern of localization of methylated histone H3 is similar to the pattern observed for Su(var)3-9, a modification that is carried out by Su(var)3-9. The antibody behaves differently and does not detect Su(var)3-9 at the chromocenter. One clear possibility is that the epitope of Su(var)3-9 to which the antibody was raised is hidden at many binding sites, including the chromocenter. Indeed this is exactly the situation that occurs with human HP1 gamma. Antibodies that recognized the amino terminal half of the protein failed to detect HP1 gamma at centric regions of chromosomes, while an antibody that recognized the carboxy end of the protein does detect HP1 gamma in centric regions of chromosomes. This issue is being addressed by preparing antibodies to other regions of Su(var)3-9 (Ner, 2002).
The data show that the pattern of hypersensitive sites over the histone genes is dramatically altered in Su(var)3-9 mutants relative to Oregon-R. For example, both the number and the relative position of the hypersensitive sites in the noncoding region between HIS1 and HIS3 genes are drastically reduced, from 10 primary sites in Oregon-R to 1 in the Su(var)3-9 mutants. The absence of smearing in the nuclease digestion pattern suggests that the change in chromatin structure observed in Su(var)3-9 mutants is homogeneous, occurring at each of the 110 copies of the histone repeat unit. While there is a dramatic alteration in nuclease hypersensitive sites, no loss of nucleosomes is observed in Su(var)3-9 mutants vs. wild type. This is interpreted to mean that the structure of the HIS-C region is altered in Su(var)3-9 mutants but the reorganized chromatin is ordered and uniform over each of the 110 his gene units. Since the number of nuclease hypersensitive sites is reduced, especially in the intergenic regions, in virtually all of the his repeat units in Su(var)3-9 strains vs. wild type it is inferred that Su(var)3-9 mutants provide a more 'open' chromatin configuration. Indeed, the data show that P-element insertions and EMS-induced mutations in Su(var)3-9 cause an increase in the steady-state levels of the histone transcripts. The increase in histone mRNA levels varies somewhat from one mutant genotype to another, as expected, but generally averages about twofold in the various Su(var)3-9 mutants examined. The strains in which the P element is precisely excised show that a return of histone transcript amounts to wild-type levels. This is the first time it has been shown that a protein modifying histone gene expression is a component of the histone chromatin domain. This is also the first demonstration that alterations in Su(var)3-9 protein are associated with alterations in both chromatin structure and gene expression at one of its target loci (Ner, 2002).
Finally, since histone gene transcription is tightly regulated and coordinated with DNA replication, one presumes that the rate of histone gene transcription must be modulated from one tissue type or developmental stage to another, commensurate with the changes in the length of S-phase. There are two ways in which this modulation in histone gene transcription could be accomplished. Either the transcription rates of all 110 copies of the his unit are modulated similarly or, alternatively, the number of his units that are actively transcribed varies with the length of the S-phase. While the Su(var)3-9 mutants cause a dramatic change in the number of nuclease hypersensitive sites, especially in the intergenic regions, the histone units were uniformly cut (no smearing). This uniformity in packaging of the histone repeat units infers that all 110 copies of the his repeats are packaged and transcribed similarly; that is, the HIS-C is modulated as a cluster. The only other hypothesis consistent with these observations is that silent his units completely lack hypersensitive sites while active his units have hypersensitive sites. But even under this scenario the Su(var)3-9 mutants must alter the hypersensitive site pattern of the actively transcribed his units. The active his units could be distinguished from the inactive units through a replication-independent deposition of the variant histone H3.3 into the nucleosomes encompassing the active units. Such a situation is observed with the regulation of transcription of the rDNA arrays. Methylated-lysine 9 histone H3 is absent in transcriptionally active loci but is present in the inactive loci (Ner, 2002).
Histones not only are essential in all eukaryotic cells, but also must be made in the correct stoichiometric ratios to serve as the basic substrates of DNA packaging. Indeed, H2A-H2B and H3-H4 genes are generally organized as pairs and often clustered in many organisms. The conservation of histone gene organization may reflect the need for, and maintenance of, this balanced expression. Strains heterozygous for a deletion of the HIS-C region, Df(2L)DS5/+, result in a three- to fivefold increase in the histone mRNA production/template. Unlike the mutations in the Su(var)3-9 gene, hemizygosity for the HIS-C region appears to uncouple the stoichiometry in histone production. Therefore, while mutations in Su(var)3-9 and hemizygosity for the HIS-C region both result in an increase in histone gene expression, the mechanisms by which this increase occurs may differ. Consistent with this hypothesis, the effect of combining Su(var)3-9 mutants with the HIS-C deletion appears to be additive, rather than epistatic or synergistic. It is not known why hemizygosity for the HIS-C region leads to an increase in histone transcripts. It is known that response to deletion of one copy of HIS-C, the HIS-C region on the normal homolog is capable of undergoing magnification. Magnification, which requires four to five generations, is not the basis of the increase in transcript levels that is reported in this study since the increase in transcript levels is observed among the F1 of outcrossed strains. It is hypothesized that the gene regulation of the HIS-C locus may be pairing sensitive and that the deletion of one copy of HIS-C disrupts pairing and leads to overexpression of the histone genes from the remaining copy. Consistent with this hypothesis, in Drosophila embryos the histone locus was found to pair more frequently and more rapidly than other euchromatic loci. The HIS-C deletion stocks and mutations in genes that influence transvection are currently being tested to see if any of these alter the chromatin structure and/or the expression of the HIS-C locus (Ner, 2002).
DNA microarray studies in Saccharomyces cerevisiae have shown that when the histone stoichiometry is altered, for example, by deletion of the yeast histone gene pair HTA2-HTB2 encoding H2A and H2B or by reduction of histone H4 levels, the expression profiles of ~10%-15% of the genetic loci are altered. Nuclei isolated from Su(var)3-9 mutants and wild-type strains were examined to determine whether the twofold increase in histone gene transcripts led to a detectable increase in the amount of histones associated with total chromatin. A doubling of the amount of histone proteins associated with chromatin was not expected, nor was it detected. However, an increase of ~20%-25% in histone H3, acetylated H4, and H1 was detected. Since all three histone types, which represented core, modified, and linker histones, respectively, were increased to similar levels, it is believed that the alteration in HIS-C expression caused by Su(var)3-9 mutations alters chromatin packaging at many areas of the genome. The 20% increase in the amount of nuclear histones that was detected in Su(var)3-9 mutants correlates reasonably well with the observation that the expression of 10%-15% of the yeast genome is altered when histone levels are altered in yeast strains. Studies in yeast have also shown that telomeric silencing, a gene-silencing phenomenon closely resembling PEV, is strongly reduced when one of two H3 and H4 gene pairs is deleted. Furthermore, mutations in HIR1 and SPT21 genes of yeast, which cause a misregulation of histone gene transcription in opposite directions, change the histone stoichiometry and result in suppression and enhancement of telomeric-position effect, respectively. While all these studies link alterations in histone amounts to altered gene expression at many loci in the genome, and in particular, to alterations in the gene silencing associated with TPEV, no mechanism has been proposed to explain these effects (Ner, 2002 and references therein).
Finally, the doubling in histone mRNA that was detected in Su(var)3-9 mutant strains does not result in a twofold increase in the amount of nucleosomes or histones associated with chromatin in these strains. Clearly, doubling the number of histones/unit of DNA is probably impossible and, if attempted, would no doubt result in lethality early in development. Since no significant reduction was detected in viability among Su(var)3-9- homozygotes, HIS-C hemizygotes, or double-mutant strains, it is assumed that there is a mechanism that prevents excess formation and/or transport of the histones to the nucleus (Ner, 2002).
While there is no evidence that directly links alterations in histone levels to PEV, the histones are key packaging components and it is curious that both hemizygosity for HIS-C and mutations of Su(var)3-9 increase histone transcript levels by about twofold. This implies that alteration in histone transcript levels can suppress PEV either directly or indirectly. But clearly the Su(var)3-9 mutants are very strong dominant suppressors of PEV, whereas HIS-C deletion lines are only moderate suppressors of PEV. However, the level to which the histone transcripts are elevated in the Su(var)3-9 mutants and in the HIS-C deletion lines is similar. This indicates that enlarging the pool of histones available to the cell at the time of DNA replication cannot be the only factor influencing the packaging and/or expression of the genes subjected to PEV. Since methylated-lysine 9 H3 is associated with heterochromatin and silenced genes, the HMTase activity of Su(var)3-9 could play a significant role in determining directly or indirectly the transcriptional competency of a variegating locus. Perhaps the decrease in methylated histone H3 gives a dramatic advantage to the formation of euchromatin, which replicates early, and concomitantly impairs the formation of heterochromatin, perhaps through the extended presence and use of the variant histone H3.3. Equally, the lack of methylation may tend to position the variegating segment of the genome in a compartment of the nucleus that is more favorable to its transcription. The role of Su(var)3-9 in PEV is currently being investigated (Ner, 2002).
In summary, it is believed that the HIS-C locus provides a good target locus for dissecting the role of various chromatin-associated proteins in both chromatin architecture and gene expression. The data show that the HIS-C region in Drosophila, and perhaps also in other organisms, is an excellent domain for examining proteins involved in establishing chromatin-domain structure and in regulating gene expression. Future experiments will be directed at examining how the entire HIS-C region is packaged into chromatin with the aim of determining the status of other histone modifications and the involvement of trans-regulators of histone gene expression (Ner, 2002).
Heterochromatin proteins are thought to play key roles in chromatin structure and gene regulation, yet very few genes have been identified that are regulated by these proteins. Large-scale mapping and analysis was performed of in vivo target loci of the proteins HP1, HP1c, and Su(var)3-9 in Drosophila Kc cells, which are of embryonic origin. For each protein, ~100-200 target genes were identified among >6000 probed loci. HP1 and Su(var)3-9 bind together to transposable elements and genes that are predominantly pericentric. In addition, Su(var)3-9 binds without HP1 to a distinct set of nonpericentric genes. On chromosome 4, HP1 binds to many genes, mostly independent of Su(var)3-9. The binding pattern of HP1c is largely different from those of HP1 and Su(var)3-9. Target genes of HP1 and Su(var)3-9 show lower expression levels in Kc cells than do nontarget genes, but not if they are located in pericentric regions. Strikingly, in pericentric regions, target genes of Su(var)3-9 and HP1 are predominantly embryo-specific genes, whereas on the chromosome arms Su(var)3-9 is preferentially associated with a set of male-specific genes. These results demonstrate that, depending on chromosomal location, the HP1 and Su(var)3-9 proteins form different complexes that associate with specific sets of developmentally coexpressed genes (Greil, 2003).
The DamID chromatin profiling technique was used to map in vivo target genes of HP1 and Su(var)3-9 in cultured D. melanogaster Kc167 cells. In brief, this technique involves in vivo expression of a trace amount of a chromatin protein of interest fused to Escherichia coli DNA adenine methyltransferase (Dam). As a result, DNA in the target loci of the chromatin protein is preferentially methylated by the tethered Dam. Subsequently, methylated DNA fragments are isolated, labeled with a fluorescent dye, and hybridized to a microarray. To correct for nonspecific binding of Dam and local differences in DNA accessibility, methylated DNA fragments of control cells transfected with Dam alone are labeled with a different fluorescent dye and cohybridized. The obtained ratio of fluorescent dyes reflects the extent of protein binding to the probed gene. The protocol was modified by replacing the purification of methylated fragments with a PCR-based selective amplification of methylated fragments. Control experiments confirm that the relative abundance of methylated sequences was conserved in the PCR amplification step. This new protocol is much more efficient and requires considerably smaller amounts (>20-fold reduction) of genomic DNA compared with the original protocol (Greil, 2003).
Based on the well documented roles of HP1 and Su(var)3-9 in the silencing of reporter genes, it was expected that the genes identified as targets would be mostly inactive. IndeedSu(var)3-9 and HP1 preferentially associate with genes of low expression levels. This preference is more prominent for Su(var)3-9 than for HP1; in fact, genes that are bound by Su(var)3-9 without HP1 are more often inactive than genes that are bound by both proteins. Formally, binding of Su(var)3-9 may be either the cause or the consequence of gene silencing. In the latter case, Su(var)3-9 complexes would mark genes that are already inactive due to other silencing mechanisms. However, if Su(var)3-9 is indeed actively involved in the silencing of its target genes, then Su(var)3-9 complexes may be more potent silencers if they lack HP1 (Greil, 2003).
Although HP1 and Su(var)3-9 generally display a preference for genes of low activity, a considerable fraction of their target genes are expressed, sometimes even at high levels. Many of these active target genes are located in pericentric regions and on chromosome 4. Earlier findings already demonstrated that the pericentric genes light and rolled are active, and it was confirmed that these genes are also bound by HP1 and Su(var)3-9. Association of HP1 was reported recently with ecdysone- and heat shock-induced puffs on polytene chromosomes. The expression of lt, rl, and hsp70 genes is reduced in HP1-deficient larvae, suggesting that HP1 may facilitate rather than suppress transcription of certain genes. Attempts were made to extend these observations in Kc cells by microarray mRNA expression profiling after RNA interference of HP1 and Su(var)3-9. No changes in expression of the HP1 or Su(var)3-9 target genes were detected after knockdown of either of the two proteins. According to these results, HP1 and Su(var)3-9 may have only redundant roles in gene regulation in Kc cells. However, it should be noted that the dsRNA-induced reduction of HP1 and Su(var)3-9, although substantial, may have been insufficient or not long enough to cause detectable alterations in gene regulation. In addition, the previously reported changes in expression of the lt and rl genes in HP1-deficient larvae were only ~2.5-fold. Such modest changes in gene expression may have been missed in a microarray-based assay. Finally, HP1 and Su(var)3-9 complexes may not be essential for gene regulation in Kc cells. Heterochromatin-mediated silencing of a reporter gene is not fully developed until late embryogenesis. Kc cells appear to be embryonic, so it is possible that the regulatory functions of HP1 and Su(var)3-9 initiate only later in development. Furthermore, the lack of a visible phenotype of Su(var)3-9 null mutants suggests that a role of Su(var)3-9 in gene regulation may be redundant (Greil, 2003).
Among the target loci of HP1 and Su(var)3-9, two conspicuous groups of developmentally coregulated genes were identified. In the first group, many of the nonpericentric genes that are exclusively bound by Su(var)3-9 in Kc cells are highly expressed in adult males, but much less in females, embryos, larvae, and Kc cells. Extrapolating these data to the entire genome [comparison of Su(var)3-9 binding and developmental expression patterns was only possible for ~2700 genes], it is anticipated that 50-100 male-specific genes are bound by Su(var)3-9 in Kc cells. This could be an underestimate, because testis-specific genes may be underrepresented in the cDNA libraries present on the microarray (Greil, 2003).
Su(var)3-9 may contribute to repression of these male-specific genes in early stages of development and in adult females. Kc cells are female, as judged from the absence of a Y chromosome and expression of the female-specific but not the male-specific splicing variant of doublesex. Therefore binding of Su(var)3-9 to these genes may reflect either the female or the embryonic origin of the Kc cells. Alternatively, aberrant expression of these genes in embryos, larvae, and female adults may not lead to a detectable phenotype under laboratory conditions (Greil, 2003).
The second group of developmentally coregulated target genes is formed by a set of embryo-specific genes. Strikingly, these embryo-specific target genes are strongly concentrated in pericentric regions, and are typically bound by both HP1 and Su(var)3-9. This suggests a specialized role for pericentric HP1 and Su(var)3-9 in the embryonic gene expression program. In Kc cells, these pericentric target genes are generally not repressed, consistent with the embryonic origin of these cells. Both HP1 and Su(var)3-9 are present in embryos, suggesting that the lack of repression of pericentric target genes cannot be attributed to the absence of either HP1 or Su(var)3-9 during this developmental stage. Rather, these proteins may facilitate gene expression in the embryo, or perhaps mark the embryonic pericentric genes for silencing later in development. The clustering of these genes in the pericentric region may play a role in their coordinated regulation (Greil, 2003).
It is likely that the genomic binding pattern of the proteins studied here depends at least in part on the cell type or developmental stage. Evidence that the target specificity of heterochromatin proteins is dynamic comes from recent observations that HP1 binds to induced but not to uninduced heat-shock and ecdysone-responsive genes. The HP1 binding map obtained in Kc cells shows only limited overlap with the banding pattern of HP1 staining on polytene chromosome arms in salivary glands. Of 91 nonpericentric target loci identified, only nine coincide with HP1 bands in polytene chromosomes. Although this comparison should be interpreted with caution because of the different methodology and a >10-fold difference in mapping resolution (the median size of the stained polytene chromosome regions is 74 kb, whereas the median size of the genomic regions probed by the microarray is 3.7 kb), it suggests that many target loci may be cell type-specific. An example of this is region 31, a broad (~0.5 Mb) region on chromosome 2 that is bound by HP1 in polytene chromosomes in salivary gland tissue. In Kc cells, three out of 50 probed loci in this region are associated with HP1, which is unlikely to account for the extensive HP1 staining of region 31 in polytene chromosomes. This suggests that much of the binding of HP1 to this region in salivary gland cells is cell type-specific (Greil, 2003).
The heterogeneity of the HP1 and Su(var)3-9 complexes, in terms of both protein composition and target gene expression status, further complicates the matter of defining heterochromatin. By morphological criteria, heterochromatin in Drosophila chromosomes is concentrated in pericentric regions. However, most pericentric genes, although bound by HP1 and Su(var)3-9, are transcriptionally active, contrary to the repressive role that is generally attributed to heterochromatin. On the 'euchromatic' chromosome arms, genes bound by Su(var)3-9 are often repressed, yet these genes typically lack the classical heterochromatin marker protein HP1. Over the long term, it may be more useful to define different types of chromatin according to their protein composition, including posttranslational modifications and histone variants. This will require a much more sophisticated nomenclature than 'euchromatin' and 'heterochromatin'. The transcriptional status of a gene may be expected to be controlled by the combinatorial action of the proteins that are associated with it. Global approaches to study chromatin composition on a gene-by-gene basis, such as described here, will be essential to catalog the different chromatin types and to understand their role in gene regulation (Greil, 2003).
HP1 is a structural component of silent chromatin at telomeres and centromeres. Euchromatic genes repositioned near heterochromatin by chromosomal rearrangements are typically silenced in an HP1-dependent manner. Silencing is thought to involve the spreading of heterochromatin proteins over the rearranged genes. HP1 associates with centric heterochromatin through an interaction with methylated lysine 9 of histone H3, a modification generated by SU(VAR)3-9. The current model for spreading of silent chromatin involves HP1-dependent recruitment of SU(VAR)3-9, resulting in the methylation of adjacent nucleosomes and association of HP1 along the chromatin fiber. To address mechanisms of silent chromatin formation and spreading, HP1 was fused to the DNA-binding domain of the E. coli lacI repressor and expressed in Drosophila melanogaster stocks carrying heat shock reporter genes positioned 1.9 and 3.7 kb downstream of lac operator repeats. Association of lacI-HP1 with the repeats results in silencing of both reporter genes and correlates with a closed chromatin structure consisting of regularly spaced nucleosomes, similar to that observed in centric heterochromatin. Chromatin immunoprecipitation experiments have demonstrated that HP1 spreads bi-directionally from the tethering site and associates with the silenced reporter transgenes. To examine mechanisms of spreading, the effects of a mutation in Su(var)3-9 were investigated. Silencing is minimally affected at 1.9 kb, but eliminated at 3.7 kb, suggesting that HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner (Danzer, 2004).
Upon daily production of the lacI-HP1 fusion protein (produced by heat shock), silencing of the reporter genes is observed at ectopic locations, even within regions of robust transcriptional activity. By contrast, a single pulse of lacI-HP1 in the embryo results in at best, partial silencing at the larval stage. This lack of mitotic stability is reminiscent of results obtained using tethered Polycomb (Pc), a protein required for the stable silencing of homeotic loci. Faithfully inherited silencing was observed with a single pulse of Gal4-Pc only when the transgene included a PRE (Polycomb Response Element), thought to stabilize the silencing complex. To date, HP1-mediated gene silencing has been shown to be relatively independent of DNA sequences; therefore, the continued presence of HP1 appears to be required for heritability of the silenced state (Danzer, 2004 and references therein).
Upon association of HP1 at these ectopic locations, changes were observed in gene expression and chromatin structure at least 3.7 kb from the lac repeat array. Sequences adjacent to the tethering site are relatively inaccessible to nuclease digestion and packaged into regular nucleosome arrays, mimicking a heterochromatic state. Such chromatin features are similar to those that form over euchromatic genes when placed into juxtaposition with heterochromatin. HP1 might cause chromatin reorganization through the recruitment of chromatin remodeling factors. An interaction between HP1 and chromatin remodeling machines has been documented in mammalian systems. Chromatin reorganization might also occur through the spread of HP1 along the chromosome. The data clearly demonstrate HP1 association within the promoter regions of silenced reporter genes up to 3.7 kb from the tethering site (Danzer, 2004).
In contrast to the silencing over several kb shown here, an HP1 tethering system using a stably integrated reporter gene in mammalian cell culture has demonstrated only short range effects over a few hundred base pairs. In this case, HP1Hsalpha was recruited to a reporter transgene through an interaction with tethered KRAB/KAP1 interaction partners. Silencing and a less accessible chromatin structure were apparent at 0.28 kb from the tethering site, but not at 2.78 kb. One possible explanation for the difference between these two tethering studies might be that human HP1Hsalpha and Drosophila HP1 have distinctly different silencing mechanisms. This is thought unlikely since human HP1Hsalpha localizes appropriately and rescues the lethality of Su(var)2-5 mutants when expressed in Drosophila. A second possibility is that lacI-HP1 overexpression enhances spreading, whereas the KRAB/KAP1 system operates under endogenous levels of HP1. A third explanation to account for the different results might be the manner in which the HP1 proteins are recruited to the reporter gene. Using the lacI-HP1 tethering system, recruitment occurs through a heterologous DNA-binding domain fused to the N terminus of HP1, thus leaving the CSD available for homodimerization and/or interaction with other partners. In the KRAB/KAP1 tethering system, recruitment occurs through an interaction between the HP1Hsalpha CSD and the transcriptional co-repressor KAP1, which may limit its availability for interactions with partners that are required for long distance spreading (Danzer, 2004 and references therein).
Transcriptional repressors can regulate gene expression over both short and long distances. Short-range repressors such as Giant and Krüppel operate at distances of less than 100 bp. These repressors frequently bind to sites within the promoter region and recruit histone deacetylases that locally deacetylate histone tails. By contrast, long-range silencing is hypothesized to involve the spread of silencing factors along the chromatin fiber, deacetylation of histone tails and generation of the MeH9K3 modification throughout the region. In experiments described here, silencing was observed 3.7 kb from the HP1 tethering site, implying that HP1 acts as a long-range silencer. Evidence of HP1 spreading is demonstrated by chromatin immunoprecipitation experiments that place HP1 near the promoter region of the silenced reporter genes. As the distance from the tethering site increases, the amount of HP1 association decreases, supporting a linear spreading model. However, these data do not exclude the possibility that HP1 association and silencing occur through a looping mechanism that is mediated by the `stickiness' of silencing proteins (Danzer, 2004 and references therein).
One proposed linear spreading model involves the association of HP1, subsequent recruitment of SU(VAR)3-9, and methylation of adjacent histones, forming new HP1-binding sites. This model was tested by examining the effects of HP1 tethering in a Su(var)3-9 mutant background. In the absence of SU(VAR)3-9, HP1 induced silencing of the hsp26 reporter persisted at 1.9 kb from the tethering site. Consistent with this finding Su(var)3-906 also had virtually no effect on silencing of a mini-white transgene positioned 0.5 kb from the HP1 tethering site. Taken together, these data suggest that silencing up to 1.9 kb is not heavily dependent upon SU(VAR)3-9 activity. It is speculated that HP1 might self-propagate for a limited distance along the chromosome, perhaps by multimerization through the CSD or by MeK9H3-independent interactions with histones. The introduction of HP1 mutants that abolish homodimerization into the tethering system will shed light on this issue (Danzer, 2004).
In contrast to the persistence of silencing at 1.9 kb in the Su(var)3-9 mutant, a substantial loss of silencing was observed at 3.7 kb. Heat shock-induced expression of hsp70 during HP1 tethering in a Su(var)3-906 mutant background was equal to expression levels observed in the non-tethering and GFP-tethering conditions. Several explanations could account for the different SU(VAR)3-9 requirements observed for silencing the hsp26 and hsp70 reporters. (1) The hsp70 transgene promoter might be stronger than the hsp26 transgene promoter -- this is thought unlikely since the hsp26 transgene appears to show greater fold induction than hsp70 at all five of the genomic insertion sites tested here under non-tethering conditions. (2) The two heat shock genes could have different mechanisms of transcriptional activation. This idea is inconsistent with years of research demonstrating that the regulatory elements and trans-activators for these two genes are nearly identical. (3) Alternatively, the differences observed might be due to multiple mechanisms of HP1-mediated silencing. Silencing at long distances (between 1.9 and 3.7 kb) may require SU(VAR)3-9, as current models for HP1 spreading would predict. By contrast, silencing at short distances (less than 1.9 kb) is relatively independent of SU(VAR)3-9, and would suggest alternate mechanisms of HP1 spreading that might involve self-propagation. This model is favored since several recent reports demonstrate that HP1 can be found independently of SU(VAR)3-9 and MeK9H3 on chromosomes. In particular, others have demonstrated that several genes silenced in Drosophila Kc cells were associated with HP1, but not SU(VAR)3-9. Thus, understanding of the role of HP1 in gene regulation will depend upon knowledge about the method of localization and the interaction partners at a given genomic site (Danzer, 2004).
Histone-tail modifications play a fundamental role in the processes that establish chromatin structure and determine gene expression. One such modification, histone methylation, was considered irreversible until the recent discovery of histone demethylases. Lsd1 was the first histone demethylase to be identified (Shi, 2004). Lsd1 is highly conserved, from yeast to humans, but its function has primarily been studied through biochemical approaches. The mammalian ortholog has been shown to demethylate monomethyl- and dimethyl-K4 and -K9 residues of histone H3. This study, along with a second study by Rudolph (2007) describes the effects of Lsd1 (Suppressor of variegation 3-3) mutation in Drosophila. The inactivation of dLsd1 strongly affects the global level of monomethyl- and dimethyl-H3-K4 methylation and results in elevated expression of a subset of genes. dLsd1 is not an essential gene, but animal viability is strongly reduced in mutant animals in a gender-specific manner. Interestingly, dLsd1 mutants are sterile and possess defects in ovary development, indicating that dLsd1 has tissue-specific functions. Mutant alleles of dLsd1 suppress positional-effect variegation, suggesting a disruption of the balance between euchromatin and heterochromatin. Taken together, these results show that dLsd1-mediated H3-K4 demethylation has a significant and specific role in Drosophila development (Di Stefano, 2007).
Su(var)3-3, the Drosophila homolog of the human LSD1 amine oxidase, demethylates H3K4me2 and H3K4me1 and facilitates subsequent H3K9 methylation by SU(VAR)3-9. Su(var)3-3 dictates the distinction between euchromatic and heterochromatic domains during early embryogenesis. Su(var)3-3 mutations suppress heterochromatic gene silencing, display elevated levels of H3K4me2, and prevent extension of H3K9me2 at pericentric heterochromatin. Su(var)3-3 colocalizes with H3K4me2 in interband regions and is abundant during embryogenesis and in syncytial blastoderm, where it appears concentrated at prospective heterochromatin during cycle 14. In embryos of Su(var)3-3/+ females, H3K4me2 accumulates in primordial germ cells, and the deregulated expansion of H3K4me2 antagonizes heterochromatic H3K9me2 in blastoderm cells. These data indicate an early developmental function for the Su(var)3-3 demethylase in controlling euchromatic and heterochromatic domains and reveal a hierarchy in which Su(var)3-3-mediated removal of activating histone marks is a prerequisite for subsequent heterochromatin formation by H3K9 methylation (Rudolph, 2007).
The homeobox (Hox) gene locus is subject to extensive H3-K4 methylation by trithorax-group proteins. It was therefore asked whether the expression level of the Hox genes Ultrabithorax (Ubx) and abdominal-A (abdA) is affected by dLsd1 depletion. Ubx- and abdA-mRNA levels increased 2-fold in SL2 cells treated with dLsd1 double-stranded RNA (dsRNA). These changes were specific and were not seen with other control genes (dDP and Hid). To verify the relevance of these observations in vivo, the expression of these genes was compared in wild-type and dLsd1ΔN mutant flies. A significant upregulation of each of these targets was found in dLsd1ΔN mutant flies, confirming the importance of dLsd1-mediated repression in vivo. Intriguingly, it was observe that this upregulation is age dependent: The difference in gene expression is minimal in larval stages, and, consistent with this, the Hox gene-expression pattern in imaginal discs from dLsd1ΔN mutant larvae and in embryos is largely unaltered. However, the level of nAcrβ, Ubx, and Abd-B gradually and significantly increases with age after eclosion, suggesting that dLsd1 function is especially important for the regulation of gene expression in adult tissues (Di Stefano, 2007).
The data support a model in which heterochromatin formation and gene silencing in PEV are defined during early embryonic development of Drosophila. A dynamic balance between HMTases and demethylases controls establishment of the functionally antagonistic histone H3K4 and H3K9 methylation marks at the border region of euchromatin and heterochromatin. In transcriptionally silent cleavage nuclei, chromatin is in a naive state with only little H3K9me2 and with H3K4 methylation completely missing. A dramatic transition of chromatin structure occurs during blastoderm formation and cellularization by establishing H3K4 and H3K9 methylation. In contrast to H3K9 acetylation, which is already found in cleavage chromatin, H3K4 methylation at prospective euchromatin appears first at the end of cleavage in cycle 12. In parallel, di- and trimethylation of H3K9 and HP1 binding establish heterochromatin. Pole cells, which are the primordial germ cells of Drosophila, are in a transcriptionally silent state and show extensive H3K9me2 and H3K9me3. During the definition of the euchromatin-heterochromatin boundaries in blastoderm cells and for the establishment of repressive H3K9 methylation marks in primordial germ cells, the SU(VAR)3-3 demethylase plays an early and inductive regulatory role. SU(VAR)3-3 might also be involved in control of early transcriptional activities within Drosophila pericentromeric sequences preceding heterochromatin formation, as suggested by a model of heterochromatin formation that depends on the RNAi pathway (Rudolph, 2007).
Genetic analysis revealed that SU(VAR)3-3 functions upstream of the H3K9 HMTase SU(VAR)3-9 and the heterochromatin-associated proteins HP1 and SU(VAR)3-7 in control of gene silencing in PEV. Combined with earlier studies of epigenetic interactions, heterochromatic gene silencing is established by a sequential action of SU(VAR)3-3, SU(VAR)3-9, the amount of Y heterochromatin, HP1, and SU(VAR)3-7. RPD3 also acts upstream of SU(VAR)3-9, because Rpd3 mutations dominate the dose-dependent PEV enhancer effect of SU(VAR)3-9. Additional genomic copies of Su(var)3-3 are epistatic to a Rpd3 mutation placing the H3K4 demethylase SU(VAR)3-3 together with RPD3 at the top of a mechanistic hierarchy controlling heterochromatic gene silencing in Drosophila. Such a role is in agreement with the enriched association of SU(VAR)3-3 to prospective heterochromatin in early blastoderm nuclei. In Su(var)3-3 null embryos, there is an extension of H3K4me2 and concomitant reduction of H3K9me3 at prospective heterochromatin, suggesting that SU(VAR)3-3 has a protective function at heterochromatic regions to restrict expansion of H3K4 methylation. Similarly, H3K9 acetylation becomes expanded toward heterochromatin. H3K4 methylation precedes H3K9 methylation in blastoderm nuclei, and both SU(VAR)3-3 and SU(VAR)3-9 are abundant proteins within cleavage chromatin. A developmentally regulated silencing complex between SU(VAR)3-3, RPD3, and SU(VAR)3-9 is therefore likely to dictate the distinction between euchromatic and heterochromatic domains during early embryogenesis. A comparable functional crosstalk between human LSD1 and HDAC1/2, which depends on nucleosomal substrates and the CoREST protein, has been demonstrated in vertebrates. The interaction between SU(VAR)3-3 and RPD3 could also explain butyrate sensitivity of Su(var)3-3 mutations. The effect of SU(VAR)3-3 on heterochromatin formation during blastoderm could involve both maternal and zygotic protein. Association of SU(VAR)3-3 with cleavage chromatin is dependent on maternal sources. In contrast, all other effects on gene silencing are zygotically determined, and no maternal effects on PEV were found in any of the Su(var)3-3 mutations. This is also supported by clonal analysis showing early onset and stable maintenance of gene silencing in PEV (Rudolph, 2007).
Modification of histones can have a dramatic impact on chromatin structure and function. Acetylation of lysines within the N-terminal tail of the histone octamer marks transcriptionally active regions of the genome whereas deacetylation seems to play a role in transcriptional silencing. The methylation of the histone tails has also been shown to be important for transcriptional regulation and chromosome structure. It is shown by immunoaffinity purification that two activities important for chromatin-mediated gene silencing, the histone methyltransferase SU(VAR)3-9 and the histone deacetylase HDAC1 (Rpd3), associate in vivo. The two activities cooperate to methylate pre-acetylated histones. Both enzymes are modifiers of position effect variegation and interact genetically in flies. A model is suggested in which the concerted histone deacetylation and methylation by a SU(VAR)3-9/HDAC1-containing complex leads to a permanent silencing of transcription in particular areas of the genome (Czermin, 2001).
This study reports the association of the HIM SU(VAR)3-9 and the deacetylase HDAC1 within Drosophila embryo extracts. This interaction plays an essential role in SU(VAR)3-9's ability to methylate acetylated histone tails, which is probably important during the 'invasion' of euchromatic regions of the genome by heterochromatin, an process observed when a gene is placed closed to heterochromatin (Czermin, 2001).
In addition to their biochemical interaction, a strong genetic interaction is seen between Su(var)3-9 and HDAC1. A point mutation within the HDAC1 gene, which has a strong Su(var) phenotype, very efficiently dominates the 'triplo-enhancer effect' usually seen in flies carrying additional copies of Su(var)3-9. The effect of different HDAC1 mutations on PEV has been described as enhancing, suppressing or neutral depending on the experimental setup. These controversial results are probably due to the different nature of the mutants used in the different laboratories. A functional redundancy of HDAC1 with other known HDACs could, for example, obscure the contribution of HDAC1 in a hypomorphic strain. It is postulated that in the HDAC1326 strain a mutant protein is made that is able to interact with SU(VAR)3-9 but fails to deacetylate the histone substrate and therefore acts as a dominant-negative suppressor (Czermin, 2001).
Based on these findings a model is proposed in which deacetylation precedes methylation of lysine 9 in the N-terminus of histone H3. Methylated H3 would then serve as a docking site for HP1, which in turn could help in assembling a specialized higher order chromatin structure. Since the turnover of methylated histones is slow in comparison to acetylation, it is suggested that histone methylation serves as a permanent epigenetic mark that freezes a particular chromatin conformation (Czermin, 2001).
The concerted action of deacetylation and methylation of lysine 9 in histone H3 could allow the generation of a permanently repressed chromatin structure within otherwise more accessible, acetylated chromatin of the early embryo. The interaction between SU(VAR)3-9 and HDAC1 is probably especially important at boundaries between eu- and hetero-chromatin and under circumstances when heterochromatin has been shown to 'invade' euchromatic regions like the white gene in the In(1)wm4h strain (Czermin, 2001).
The existence of a complex containing a HDAC and a HIM provides a molecular mechanism for the close connection between histone deacetylation and histone methylation. This link between deacetylation and methylation is also evident in S. pombe, where inhibition of deacetylases by TSA as well as mutations of the Su(var)3-9 ortholog clr4 leads to defects in centromere function. It will be very interesting to see whether the coupling of histone deacetylation and histone methylation is a mechanism commonly used to stably repress gene expression not only around the centromere but also in euchromatic regions of the genome (Czermin, 2001).
Su(var)3-9 is a dominant modifier of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologs, Su(var) 3-9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3-9 null mutants, H3-K9 methylation at chromocenter heterochromatin is strongly reduced, indicating that Su(var)3- 9 is the major heterochromatin-specific HMTase in Drosophila. Su(var)3-9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, Su(var)3-7. Notably, interaction between Su(var)39 and Hp1 is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of Hp1 at the chromocenter is nearly lost without affecting Hp1 accumulation at the fourth chromosome. By contrast, in Hp1 null mutants, Su(var)3-9 is no longer restricted at heterochromatin but broadly disperses across the chromosomes. Despite this interdependence, Su(var)3-9 dominates the PEV modifier effects of Hp1 and Su(var)3-7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3-9 silencing defects. Together, these data indicate a central role for the SU(VAR)3- 9 HMTase in heterochromatin-induced gene silencing in Drosophila (Schotta, 2002).
In Drosophila, histone H3-K9 methylation is strongly enriched in chromocenter heterochromatin and the fourth chromosome. Immunocytological studies revealed that SU(VAR)3-9 preferentially causes H3-K9 methylation within chromocenter heterochromatin. Although these results suggest a significant role of H3-K9 methylation in altering chromatin structure and gene activity during development, further studies are required to understand how integral components of higher order chromatin complexes in heterochromatin are assembled and their function is regulated (Schotta, 2002).
Su(var)3-9 and Hp1 represent evolutionarily conserved components of heterochromatin protein complexes. Interaction between the two proteins has been suggested for the mammalian homologs. In Drosophila, the N-terminus of Su(var)3-9 and the chromo-shadow domain region of Hp1 constitute the sites where these proteins interact. Ectopic association of Su(var)3-9-EGFP along euchromatic regions in Hp1-deficient salivary gland nuclei and strongly reduced binding of Hp1- EGFP to chromocenter heterochromatin in Su(var)3-9-deficient nuclei suggest that interaction between both proteins is essential for their association with chromocenter heterochromatin. H3-K9 methylation creates chromodomain-dependent binding sites of Hp1. Strong reduction of Hp1-EGFP heterochromatin binding in Su(var)3-9 null mutants might reflect a requirement of Hp1 binding to methylated H3-K9 for heterochromatin-association of Su(var)3-9- Hp1 complexes. These results suggest a multistep control for heterochromatin association of Su(var)3-9-Hp1 complexes. After primary association of Su(var)3-9 with heterochromatin, consecutive H3-K9 methylation by Su(var)3-9 would create binding sites of Hp1, which finally results in stable association of Su(var)3-9-Hp1 complexes with heterochromatin. These processes are likely to be controlled by several other as yet unknown factors. In these processes the chromodomain as well as the SET domain of Su(var)3-9 might be directly involved. Fusion proteins deleting either the chromodomain or the SET domain only show restricted binding to heterochromatin (Schotta, 2002).
Although Su(var)3-9 associates with the fourth chromosome, H3-K9 methylation in the fourth chromosome is not changed in Su(var)3-9 null mutants, suggesting that H3-K9 methylation in this chromosome is controlled by a different HMTase activity. In contrast to Su(var)3-9 association with chromocenter heterochromatin, which depends on the chromodomain and the SET domain, for its binding to the fourth chromosome the N-terminus is sufficient. A special chromatin structure of the fourth chromosome is also indicated by identification of Painting of fourth (Pof), a chromosome four-specific protein. Different requirements of Su(var)3-9 and Hp1 association with the fourth chromosome and chromocenter heterochromatin suggest occurrence of heterochromatin protein complexes of different composition, as well as differential control of their assembly (Schotta, 2002).
Structure-function analysis with transgenic Su(var)3-9-EGFP protein variants reveals new aspects of their role in heterochromatin localization of Su(var)3-9. In contrast to studies with human SUV39H1, in vivo heterochromatin association of Su(var)3-9-EGFP protein variants was analyzed in nuclei deficient for the endogenous Su(var)3-9 protein. The N-terminus of Su(var)3-9 (amino acids 81-188), which contains the interaction domain to Hp1 and Su(var)3-7, is involved in heterochromatin association of the protein. However, association of the truncated protein is restricted to the fourth chromosome and the central region of chromocenter heterochromatin. Deletion of the chromodomain in Su(var)3-9 also affects its normal chromosomal distribution and reduces binding to chromocenter heterochromatin, but not with the fourth chromosome. In contrast, deletion or point mutations of the chromodomain result in ectopic distribution of human SUV39H1 in HeLa cells. These findings might indicate functional differences between the Su(var)3-9 and SUV39H1 chromodomain. In both Su(var)3-9 and SUV39H1, the N-terminus contains the interaction surface for Hp1 and Hp1ß, respectively. In human cells, overexpression of SUV39H1 results in ectopic chromosomal distribution. In contrast, even after strong overexpression of Su(var)3-9, no comparable effects were observed in Drosophila (Schotta, 2002).
Deletion of the SET domain or an exchange of the Su(var)3-9 SET domain with the SET domain of the Trx protein strongly affects heterochromatin distribution of the proteins. The proteins become concentrated within the middle of chromocenter heterochromatin, but again show normal association with the fourth chromosome. This suggests that the SET domain of Su(var)3-9 is directly involved in the control of Su(var)3-9 association with chromocenter heterochromatin. In Drosophila, aberrant heterochromatin distribution of Su(var)3-9 proteins with SET domain mutations could be causally connected with suppression of heterochromatin-induced gene silencing. Comparable results have been obtained for clr4, the Schizosaccharomyces pombe homolog of Su(var)3-9, where mutations in the SET domain show defects in silencing and mating-type switching. However, in S.pombe swi6, the homolog of Su(var)2-5 represents the main dosage-dependent component of gene silencing at the mat2/3 locus, whereas only subtle effects of clr4 are reported. These functional differences observed for Su(var)3-9 and Hp1 orthologs in fission yeast, Drosophila and mammals might reflect considerable functional and/or structural differences of the silencing complexes in these organisms (Schotta, 2002).
Aberrant heterochromatin distribution of Su(var)3-9 SET domain mutant proteins suggests involvement of other factors in a functional control of the SET domain. These factors might also affect its HMTase activity. Mutations in genes encoding these putative regulatory genes should be genetically epistatic to the triplo-dependent enhancer effect of Su(var)3-9. Proteins like the SET domain-binding factor Sbf1, which has been shown to be involved in regulation of the phosphorylation state of the SET domain, might also play a central role. Identification of PEV enhancer mutations like ptn D (see pitkin) that cause ectopic binding of Su(var)3-9 and Hp1 to many euchromatic sites indicates the existence of different positive as well as negative control mechanisms for chromosomal distribution of heterochromatin protein complexes. Further studies of modifiers of PEV mutations will contribute substantially to understanding of the complex regulatory processes involved in the control of higher order chromatin structure and heterochromatin-induced gene silencing (Schotta, 2002).
In most eukaryotes, the histone methyltransferase SU(VAR)3-9 and its orthologues play a major role in the function of centromeric heterochromatin. Although the methyltransferase domain is required for the formation of a fully functional centromere, mutations within other regions of the gene such as the N-terminus also have a strong impact on its in vivo function. To analyze the contribution of the N-terminus on the methyltransferase activity, the full-length Drosophila SU(VAR)3-9 (dSU(VAR)3-9) was expressed, together with various N-terminal deletions, in Escherichia coli and the structural and enzymatic properties of the purified recombinant enzymes were analyzed. Full-length Drosophila SU(VAR)3-9 specifically methylates lysine 9 within histone H3 on peptides, on intact histones, and, to a lesser extent, on nucleosomes. A detailed analysis of the reaction products shows that SU(VAR)3-9 adds two methyl groups to an unmethylated H3 tail peptide in a nonprocessive manner. The full-length enzyme elutes with an apparent molecular weight of 160 kDa from a gel filtration column: this indicates the formation of a dimer. This property is dependent on an intact N-terminus. In contrast to the full-length enzymes, proteins lacking the N-terminus fail to dimerize, and show a 10-fold lower specific activity and a linear dependence of methyltransferase activity on enzyme concentration. An N-terminal peptide containing amino acids 1-152 of dSU(VAR)3-9 is sufficient to mediate this interaction in vitro. The dimerization of dSU(VAR)3-9 and the subsequent increase of its methyltransferase activity provide a starting point to understand the molecular details of the formation of heterochromatic structures in vivo (Eskeland, 2004).
See the embryonic expression pattern of Su(var)3-9 at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
Modifier mutations of position-effect variegation (PEV) represent a useful tool for a genetic and molecular dissection of genes connected with chromatin regulation in Drosophila. The Su(var)3-9 gene belongs to the group of haplo suppressor loci that manifest a triplo enhancer effect. Mutations show a strong suppressor effect even in the presence of PEV enhancer mutations, indicating a central role of this gene in the regulation of PEV (Tschiersch, 1994).
Histone lysine methylation is an epigenetic mark to index chromosomal subdomains. In Drosophila, H3-K9 di- and trimethylation is mainly controlled by the heterochromatic SU(VAR)3-9 HMTase, a major regulator of position-effect variegation (PEV). In contrast, H3-K27 methylation states are independently mediated by the Pc-group enzyme E(Z). Isolation of 19 point mutants demonstrates that the silencing potential of Su(var)3-9 increases with its associated HMTase activity. A hyperactive Su(var)3-9 mutant, pitkinD, displays extensive H3-K9 di- and trimethylation within but also outside pericentric heterochromatin. Notably, mutations in a novel Su(var) gene, Su(var)3-1, severely restrict Su(var)3-9-mediated gene silencing. Su(var)3-1 was identified as an'antimorphic' mutant of the euchromatic H3-S10 kinase JIL-1. JIL-1Su(var)3-1 mutants maintain kinase activity and do not detectably impair repressive histone lysine methylation marks. However, analyses with seven different PEV rearrangements demonstrate a general role of JIL-1Su(var)3-1 in controlling heterochromatin compaction and expansion. These data provide evidence for a dynamic balance between heterochromatin and euchromatin, and define two distinct mechanisms for Su(var) gene function. Whereas the majority of Su(var)s encode inherent components of heterochromatin that can establish repressive chromatin structures [intrinsic Su(var)s], Su(var)3-1 reflects gain-of-function mutants of a euchromatic component that antagonize the expansion of heterochromatic subdomains [acquired Su(var)s] (Ebert, 2004).
The identification of Su(var) genes in Drosophila reveals many important proteins regulating higher-order chromatin structure. SU(VAR)3-9, HP1, and SU(VAR)3-7 are inherent components of heterochromatin that can establish and maintain a heterochromatic chromatin structure. By genetic means, Su(var)3-9 is the dominant component over Su(var)2-5 (HP1) and Su(var)3-7, indicating a major function of the associated HMTase in the formation of repressive chromatin regions (Ebert, 2004 and reference therein).
In Drosophila, heterochromatic gene silencing is mainly determined by H3-K9 dimethylation. In agreement with its in vitro HMTase activity, Su(var)3-9 regulates H3-K9 di- and trimethylation at pericentric heterochromatin. In addition, other H3-K9-specific HMTases must exist that mediate H3-K9 dimethylation at the fourth chromosome and in the chromocenter core of Su(var)3-9 mutants. HP1 is another important control factor for H3-K9 methylation because in Su(var)2-5 mutants, H3-K9 mono- and dimethylation are dramatically increased along chromosomal arms. Although SU(VAR)3-9 shows extensive association with heterochromatic and euchromatic regions in HP1-null cells, the shift in these methylation patterns could also be mediated by other H3-K9-specific HMTases. At pericentric heterochromatin, H3-K9 methylation states appear unaltered; however, a slight reduction of H3-K9 dimethylation that is not detectable by immunofluorescence might be the cause of the HP1 Su(var) effect. Alternatively, impairment of H4-K20 trimethylation at pericentric heterochromatin could cause the loss of silencing in HP1 mutants (Ebert, 2004).
The analysis of Su(var)3-9 mutants has revealed novel hypomorphic and null alleles that show differential effects on H3-K9 methylation states. A direct correlation between their HMTase activities and the amount of gene silencing demonstrates that establishment of heterochromatic structures is to a large extent determined by the kinetic properties of the SU(VAR)3-9 HMTase reaction. Consistently, hyperactive SU(VAR)3-9ptn induces enhanced H3-K9 di- and tri-methylation, concomitant with an expansion of heterochromatin. This results in severe phenotypes such as dominant female sterility caused by overcompaction of the whole chromatin in early embryos. In this study it is shown that Su(var)3-9ptn mutants display around 100-200 additional bands with enhanced H3-K9 di- and tri-methylation in the arms of polytene chromosomes. These bands likely correspond to endogenous SU(VAR)3-9-binding sites and demonstrate that hyperactive SU(VAR)3-9ptn can mediate the expansion of repressive structures also at chromosomal arms (Ebert, 2004).
A loss-of-function Su(var) mutant reduces gene silencing by removing one or several intrinsic components of heterochromatin. Su(var)3-9ptn induces enhanced gene silencing, and it was therefore surprising to encode a mutant of a 'classical' Su(var) gene. Based on functional characterization, this mutant was identified as the first hypermorphic (gain-of-function) allele of an intrinsic Su(var) gene. Many more PEV modifier mutants that cause enhanced gene silencing have been isolated. Molecular characterization of some of these genes have revealed components of active chromatin, such as transcription factors; however, further characterization of this class of genes might also uncover other hypermorphic mutants of intrinsic Su(var) genes and contribute to the understanding of heterochromatin regulation (Ebert, 2004).
The mutant screen to identify genes that impair Su(var)3-9-mediated gene silencing led to the molecular identification of Su(var)3-1, the strongest Su(var) gene to date. Su(var)3-1 can restrict heterochromatin formation and generally dominate over the strong silencing effect of Su(var)3-9 extra copies. Su(var)3-1 codes for C-terminal truncations of the H3-S10 kinase JIL-1 that does not affect the JIL-1 ability to phosphorylate H3-S10 but selectively impairs the expansion of heterochromatin (Ebert, 2004).
Intrinsic Su(var) genes impair or weaken the heterochromatic structure. Loss-of-function Su(var)3-9 alleles or mutations in HDAC1 reduce H3-K9 dimethylation, whereas HP1 mutants impair H4-K20 trimethylation at pericentric heterochromatin. JIL-1 is not an intrinsic component of heterochromatin and does not affect any of the known repressive histone lysine methylation marks at pericentric heterochromatin. Nevertheless, JIL-1Su(var)3-1 alleles display a strong Su(var) effect. Therefore JIL-1Su(var)3-1 is categorized as a novel type of mutant with acquired Su(var) function. In contrast to intrinsic Su(var)s that encode components directly involved in heterochromatin formation, acquired Su(var) mutants would antagonize heterochromatin formation by stabilizing the euchromatic state or preventing heterochromatin expansion (Ebert, 2004).
The data assign a novel function to the C terminus of the JIL-1 kinase that is independent of its H3-S10 kinase activity. Lack of the C terminus could prevent phosphorylation or interaction with an as-yet-unknown target protein. Alternatively, the C terminus could comprise control functions that prevent excessive phosphorylation of this target. There are many possible target proteins that could be involved in higher-order chromatin formation. For example, the striking X-chromosome decondensation phenotype of JIL-1Su(var)3-1 mutants has also been described for iswi and nurf301, subunits of the NURF complex. Particularly, nurf301 mutants display a dominant Su(var) effect, indicating that chromatin remodeling processes are involved in the regulation of heterochromatin. Another possible target of the JIL-1Su(var)3-1 kinase might be the histone variant H2A.Z. In budding yeast, H2A.Z is required to prevent spreading of heterochromatin into euchromatin. This protective function might be modulated by JIL-1Su(var)3-1-mediated phosphorylation of H2A.Z. Finally, some intrinsic Su(var)s such as SU(VAR)3-9 and HP1 are phosphoproteins, and JIL-1Su(var)3-1-mediated phosphorylation of those proteins might affect their function in heterochromatin expansion (Ebert, 2004).
Based on these data, a model is proposed for a dynamic balance between euchromatin and heterochromatin. In PEV rearrangements, the boundary between these two states is determined by the antagonistic functions of euchromatic regulators (e.g., JIL-1) and the SU(VAR)3-9 HMTase that mediates H3-K9 dimethylation, the major mark of heterochromatin. The boundary between euchromatin and heterochromatin is not static and depends on the activity and abundance of inherent components of heterochromatin, such as, for example, hyperactive SU(VAR)3-9ptn or overexpression of Su(var)3-9. In contrast, hypoactive or null mutants of Su(var)3-9 weaken heterochromatin formation and favor the propagation of the euchromatic state. In JIL-1Su(var)3-1 mutants, heterochromatin expansion is severely repressed, even under conditions of elevated Su(var)3-9 function, and is most likely antagonized by currently unknown proteins that are potential targets of the gain-of-function JIL-1Su(var)3-1 kinase. Dependent on the phosphorylation state of these targets, either heterochromatin expansion can be blocked or the propagation of the euchromatic state is highly stabilized (Ebert, 2004).
This work significantly extends mechanistic insights into the formation of heterochromatin, and Su(var)3-1 was discovered as a novel class of PEV modifiers that controls the balance between euchromatic and heterochromatic subdomains. Because of the dominant role of JIL-1Su(var)3-1 over major components of heterochromatin [intrinsic Su(var)s], the putative JIL-1 mutant targets are predicted to have important functions in gene silencing and the higher-order structuring of chromatin. The ongoing analysis on the full definition of Su(var) gene function is therefore likely to reveal both the genetic and molecular hierarchy that dictates epigenetic gene control (Ebert, 2004).
Cancer is both a genetic and an epigenetic disease. Inactivation of tumour-suppressor genes by epigenetic changes is frequently observed in human cancers, particularly as a result of the modifications of histones and DNA methylation. It is therefore important to understand how these damaging changes might come about. By studying tumorigenesis in the Drosophila eye, two Polycomb group epigenetic silencers, Pipsqueak and Lola, have been identified that participate in this process. When coupled with overexpression of Delta, deregulation of the expression of Pipsqueak and Lola induces the formation of metastatic tumours. This phenotype depends on the histone-modifying enzymes Rpd3 (a histone deacetylase), Su(var)3-9 and E(z), as well as on the chromodomain protein Polycomb. Expression of the gene Retinoblastoma-family protein (Rbf ) is downregulated in these tumours and, indeed, this downregulation is associated with DNA hypermethylation. Together, these results establish a mechanism that links the Notch-Delta pathway, epigenetic silencing pathways and cell-cycle control in the process of tumorigenesis (Ferres-Marco, 2006).
H3K4 methylation is thought to be permissive for maintaining and propagating activated chromatin and is thought to neutralize repressor tags by precluding binding of the HDAC complex and impairing SUV39H1-mediated H3K9 methylation. Thus, H3K4me3 depletion may contribute to tumour formation by permitting aberrant chromatin silencing. It was found that a 50% reduction in dosage of the HDAC gene Rpd3 or of Su(var)3-9 decreased the tumour phenotype dominantly. In contrast, reducing the activity of the H3K4 histone methyltransferase genes Trx (known as ALL1 or MLL in humans) or Ash1, which would be expected to deplete the H3K4me3 tag further, did not visibly enhance the tumours (Ferres-Marco, 2006).
E(z) when complexed with the Extra sex combs (Esc) protein becomes a histone methyltransferase. The E(z)-Esc complex and its mammalian counterpart Ezh2-Eed show specificity for H3K27 but may also target H3K9. The complex also contains the HDAC Rpd3, and this association with Rpd3 is conserved in mammals. H3K27 methylation facilitates binding of the chromodomain protein Pc (HPC in humans), which then creates a repressive chromatin state that is a stable silencer of genes (Ferres-Marco, 2006).
Although loss of E(z) does not cause proliferation defects within discs, halving the E(z) gene dosage dominantly suppressed tumorigenesis, indicating that histone methylation by the E(z)-Esc complex is also a prerequisite for the excessive proliferation of these tumours. Accordingly, Esc- or Pc- mutations also notably reduced the tumours (Ferres-Marco, 2006).
Together, these findings suggest that the development of these tumours involves, at least in part, changes in the structure of chromatin brought about by covalent modifications of histones. These changes probably switch the target genes from the active H3K4me3 state to a deacetylated H3K9 and H3K27 methylation silent state (Ferres-Marco, 2006).
A reduction in the levels of the JIL-1 histone H3S10 kinase results in the spreading of the major heterochromatin markers dimethyl H3K9 and HP1 to ectopic locations on the chromosome arms, with the most pronounced increase on the X chromosomes. Genetic interaction assays demonstrated that JIL-1 functions in vivo in a pathway that includes Su(var)3-9, a major catalyst for dimethylation of the histone H3K9 residue, HP1 recruitment, and the formation of silenced heterochromatin. Evidence is provided that JIL-1 activity and localization are not affected by the absence of Su(var)3-9 activity, suggesting that JIL-1 is upstream of Su(var)3-9 in the pathway. Based on these findings, a model is proposed where JIL-1 kinase activity functions to maintain euchromatic regions by antagonizing Su(var)3-9-mediated heterochromatization (Zhang, 2006: full text of article).
According to the histone code hypothesis and the recently proposed binary switch model, phosphorylation of a site adjacent to a methyl mark that engages an effector molecule may regulate its binding. JIL-1 phosphorylates the histone H3S10 residue in euchromatic regions of polytene chromosomes, raising the possibility that this phosphorylation at interphase prevents the recruitment of Su(var)3-9 and/or the dimethylation of the neighboring K9 residue. This, in turn, would affect the binding of HP1, thus antagonizing the formation of silenced heterochromatin at interbands. That different regions of chromatin may have different combinations of posttranslational modifications controlling effector/histone interactions, as predicted by the histone code hypothesis, is underscored by the finding that, in JIL-1 null backgrounds, the level of the dmH3K9 marker and HP1 are preferentially increased on the male and female X chromosomes. It is well documented that the male X chromosome is unique because of the activity of the MSL dosage compensation complex and the MOF histone acetyltransferase, which leads to hyperacetylation of histone H4. However, comparable markers for the female X chromosome have yet to be discovered, and the results are the first indication that markers may exist that distinguish male and female X chromosomes from autosomes, and that this difference may increase the affinity for Su(var)3-9. That the spreading of heterochromatic markers in the absence of JIL-1 occurs on both the male and female X chromosome further indicates that these changes are independent of dosage compensation processes (Zhang, 2006).
Unfortunately, in this study, the possibility that the observed spreading of heterochromatin markers occurred preferentially to specific euchromatic sites could not directly addressed. In JIL-1 null and hypomorphic backgrounds, chromosome morphology is greatly perturbed, and there is an intermixing not only of euchromatin and the compacted chromatin characteristic of banded regions, but also of non-homologous chromatid regions, which become fused and confluent. Thus, an alternative hypothesis that JIL-1 activity may regulate boundary elements that control the spreading of heterochromatic factors cannot be ruled out, or that the two mechanisms may act in concert. However, the spreading of the dmH3K9 marker and HP1 to ectopic locations on the chromosomes is likely to lead to heterochromatization and repression of gene expression at these sites. The results further suggest the possibility that the lethality of JIL-1 null mutants may be due to the repression of essential genes at these ectopic sites as a consequence of the spreading of Su(var)3-9 activity. This hypothesis is supported by genetic interaction assays that demonstrated that the lethality of a severely hypomorphic JIL-1 heteroallelic combination could be almost completely rescued by a reduction in Su(var)3-9 dosage that prevented the ectopic dimethylation of histone H3K9 (Zhang, 2006).
The Su(var)3-1 alleles of JIL-1 consist of dominant gain-of-function alleles that antagonize the expansion of heterochromatin formation. However, evidence is provided that the underlying molecular mechanism of this antagonism is different from that occurring in the loss-of-function null and hypomorphic JIL-1 alleles described in this study. JIL-1Su(var)3-1 alleles are characterized by deletions of the COOH-terminal domain that do not affect JIL-1 kinase activity or the spreading of heterochromatin markers. Furthermore, the results indicate that the COOH-terminal domain of JIL-1 is required for proper chromosomal localization and that JIL-1Su(var)3-1 proteins are mislocalized to ectopic chromosome sites. Thus, it is proposed that the dominant gain-of-function effect of the JIL-1Su(var)3-1 alleles may be attributable to JIL-1 kinase activity at ectopic locations, possibly through the phosphorylation of novel target proteins, or by mis-regulated localization of the phosphorylated histone H3S10 marker. Although the JIL-1Su(var)3-1 proteins are mislocalized, they still associate with chromosomes and phosphorylate the histone H3S10 residue, suggesting that other regions of the protein have a binding affinity for at least some of the substrates and interaction partners of JIL-1. This is supported by the finding that each of the two kinase domains of JIL-1 can interact with the MSL-complex in vitro (Zhang, 2006).
In summary, evidence is provided that the JIL-1 kinase is a major regulator of histone modifications that affect gene activation, gene silencing and chromatin structure. Thus, it will be informative in future experiments to further explore the interaction of JIL-1 with genes controlling heterochromatin formation, in order to gain a better understanding of the molecular mechanisms of epigenetic gene regulation (Zhang, 2006).
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date revised: 10 April 2008
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