Suppressor of variegation 3-9

Targets of Activity

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 39D_2-3-39E_1-2. 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).

Mechanisms of HP1-mediated gene silencing in Drosophila: HP1-mediated silencing can operate in a SU(VAR)3-9-independent and -dependent manner

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, HP1^Hsalpha 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 HP1^Hsalpha and Drosophila HP1 have distinctly different silencing mechanisms. This is thought unlikely since human HP1^Hsalpha 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 HP1^Hsalpha 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).

Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3

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 dLsd1^ΔN mutant flies. A significant upregulation of each of these targets was found in dLsd1^ΔN 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 dLsd1^ΔN 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 (see Drosophila 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).

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).

Irradiation responsiveness appears to be a highly conserved feature of reaper-like IAP antagonists. A recently identified functional ortholog of reaper in mosquito genomes, michelob_x (mx), is also responsive to irradiation. These results highlighted that stress responsiveness is an essential aspect of functional regulation of upstream proapoptotic genes such as reaper/hid. It is also worth mentioning that several mammalian BH3 domain-only proteins, the upstream proapoptotic regulators of the Bcl-2/Ced-9 pathway, are also regulated at the transcriptional level (Zhang, 2008).

This study shows that the irradiation responsiveness of reaper and hid is subject to epigenetic regulation during development. The epigenetic regulation of the IRER is fundamentally different from the silencing of homeotic genes in that the change of DNA accessibility is limited to the enhancer region while the promoter of the proapoptotic genes remains open. Thus, it seems more appropriate to refer this as the 'blocking' of the enhancer region instead of the 'silencing' of the gene. This region, containing the putative P53RE and other essential enhancer elements, is required for mediating irradiation responsiveness. ChIP analysis indicates that histones in this enhancer region are quickly trimethylated at both H3K9 and H3K27 at the sensitive-to-resistant transition period, accompanied by a significant decrease in DNA accessibility. DNA accessibility in the putative P53RE locus (18,368k), when measured by the DNase I sensitivity assay, did not show significant decrease until sometime after the transition period. It is possible that other enhancer elements, in the core of IRER_left, are also required for radiation responsiveness. An alternative explanation is that the strong and rapid trimethylation of H3K27 and association of PRC1 at 18,366,000-18, 368,000 are sufficient to disrupt DmP53 binding and/or interaction with the Pol II complex even though the region remains relatively sensitive to DNase I. Eventually, the whole IRER is closed with the exception of an open island around 18,387,000 (Zhang, 2008).

The finding that epigenetic regulation of the enhancer region of proapoptotic genes controls sensitivity to irradiation-induced cell death may have implications in clinical applications involving ionizing irradiation. It suggests that applying drugs that modulate epigenetic silencing may help increase the efficacy of radiation therapy. It also remains to be seen whether the hypersensitivity of some tumors to irradiation is due to the dedifferentiation and reversal of epigenetic blocking in cancer cells. In contrast, loss of proper stress response to cellular damage is implicated in tumorigenesis. The fact that the formation of heterochromatin in the sensitizing enhancer region of proapoptotic genes is sufficient to convey resistance to stress-induced cell death suggests it could contribute to tumorigenesis. In addition, it could also be the underlying mechanism of tumor cells' evading irradiation-induced cell death. This is a likely scenario given that it has been well documented that oncogenes such as Rb and PML-RAR fusion protein cause the formation of heterochromatin through recruiting of a human ortholog of Su(v)3-9. In this regard, the reaper locus, especially the IRER, provides an excellent genetic model system for understanding the cis- and trans-acting mechanisms controlling the formation of heterochromatin associated with cellular differentiation and tumorigenesis (Zhang, 2008).

The developmental consequence of epigenetic regulation of the IRER is the tuning down (off) of the responsiveness of the proapoptotic genes, thus decreasing cellular sensitivity to stresses such as DNA damage. Epigenetic blocking of the IRER corresponds to the end of major mitotic waves when most cells begin to differentiate. Similar transitions were noticed in mammalian systems. For instance, proliferating neural precursor cells are extremely sensitive to irradiation-induced cell death while differentiating/differentiated neurons become resistant to γ-ray irradiation, even though the same level of DNA damage was inflicted by the irradiation. These findings here suggest that such a dramatic transition of radiation sensitivity could be achieved by epigenetic blocking of sensitizing enhancers (Zhang, 2008).

Later in Drosophila development, around the time of pupae formation, the organism becomes sensitive to irradiation again, with LD₅₀ values similar to what was observed for the 4–7 hr AEL embryos. Interestingly, it has also been found that during this period, the highly proliferative imaginal discs are sensitive to irradiation-induced apoptosis, which is mediated by the induction of reaper and hid through P53 and Chk2. However, it remains to be studied whether the reemergence of sensitive tissue is due to reversal of the epigenetic blocking in the IRER or the proliferation of undifferentiated stem cells that have an unblocked IRER (Zhang, 2008).

The blocking of the IRER differs fundamentally from the silencing of homeotic genes in several aspects. (1) The change of DNA accessibility and histone modification is largely limited to the enhancer region. The promoter regions of reaper (and hid) remain open, allowing the gene to be responsive to other stimuli. Indeed, there are a few cells in the central nervous system that could be detected as expressing reaper long after the sensitive-to-resistant transition. Even more cells in the late-stage embryo can be found having hid expression. Yet, the irradiation responsiveness of the two genes is completely suppressed in most if not all cells, transforming the tissues into a radiation-resistant state (Zhang, 2008).

(2) The histone modification of the IRER has a mixture of features associated with pericentromeric heterochromatin formation and canonic PcG-mediated silencing. Both H3K9 and H3K27 are trimethylated in the IRER. Both HP1, the signature binding protein of the pericentromeric heterochromatin, and PRC1 are bound to the IRER. As demonstrated by genetic analysis, the functions of both Su(var)3-9 and Su(z)12/Pc are required for the silencing. Preliminary attempts to verify specific binding of PRC2 proteins to this region were unsuccessful. The fact that none of the mutants tested could completely block the transition seems to suggest that there is a redundancy of the two pathways in modifying/blocking the IRER. It is also possible that the genes tested are not the key regulators of IRER blocking but only have participatory roles in the process (Zhang, 2008).

(3) Within the IRER, there is a small region around 18,386,000 to 18,188,000 that remains relatively open until the end of embryogenesis. Interestingly, this open region is flanked by two putative noncoding RNA transcripts represented by EST sequences. If they are indeed transcribed in the embryo as suggested by the mRNA source of the cDNA library, then the 'open island' within the closed IRER will likely be their shared enhancer/promoter region. Sequences of both cDNAs revealed that there is no intron or reputable open reading frame in either sequence. Despite repeated efforts, their expression was not confirmed via ISH or northern analysis. Overexpression of either cDNA using an expression construct also failed to show any effect on reaper/hid-induced cell death in S2 cells. Yet, sections of the two noncoding RNAs are strongly conserved in divergent Drosophila genomes. The potential role of these two noncoding RNAs in mediating reaper/hid expression and/or blocking of the IRER remains to be studied (Zhang, 2008).

HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster

Heterochromatin protein 1a (HP1a) is a chromatin-associated protein important for the formation and maintenance of heterochromatin. In Drosophila, the two histone methyltransferases SETDB1 and Su(var)3-9 mediate H3K9 methylation marks that initiates the establishment and spreading of HP1a-enriched chromatin. Although HP1a is generally regarded as a factor that represses gene transcription, several reports have linked HP1a binding to active genes, and in some cases, it has been shown to stimulate transcriptional activity. To clarify the function of HP1a in transcription regulation and its association with Su(var)3-9, SETDB1 and the chromosome 4-specific protein Painting of fourth (POF), genome-wide expression studies were performed and the results were combined with available binding data in Drosophila melanogaster. The results suggest that HP1a, SETDB1 and Su(var)3-9 repress genes on chromosome 4, where non-ubiquitously expressed genes are preferentially targeted, and stimulate genes in pericentromeric regions. Further, it was shown that on chromosome 4, Su(var)3-9, SETDB1 and HP1a target the same genes. In addition, it was found that transposons are repressed by HP1a and Su(var)3-9 and that the binding level and expression effects of HP1a are affected by gene length. The results indicate that genes have adapted to be properly expressed in their local chromatin environment (Lundberg, 2013).

Heterochromatin protein 1 is a protein that has been well-studied in many model organisms, including Schizosaccharomyces pombe, mouse and D. melanogaster. Although D. melanogaster HP1a is best known for its role in heterochromatin formation and silencing, several reports have linked HP1a to regulation of transcriptional activity of heterochromatic and some euchromatic genes. This study asked if these conflicting results could partly be explained by a region-specific function of HP1a and the proteins involved in HP1a binding, i.e. SETDB1, Su(var)3-9 and POF. Based on polytene chromosome staining, it was clear that POF, HP1a and SETDB1 overlapped on chromosome 4 but not on the pericentromeric section or on the most distal part of the tip, which was only bound by HP1a. These POF and SETDB1 unbound regions also correspond to regions that are independent of SETDB1 for maintaining a proper H3K9me2 and me3 pattern. In line with previous studies, it was found that Su(var)3-9 binds to chromosome 4 when considering expressed genes, and more interestingly, this binding to active genes is, on average, stronger than the binding of Su(var)3-9 to active genes in the pericentromeric regions, although loss of Su(var)3-9 had minor effects on the methylation pattern of chromosome 4. The putative function of Su(var)3-9 on chromosome 4 therefore remains elusive (Lundberg, 2013).

In addition to the persistent binding of POF to chromosome 4, it is interesting to note the presence of occasional binding to region 2L:31. It is known that POF binds to HP1a binding sites where HP1a binding is dependent on SETDB1, and since it has been previously shown that binding of HP1a in region 2L:31 is dependent on SETDB1, this could partially explain the sporadic binding of POF in this region. Region 2L:31 displayed similar properties to other euchromatic regions that are unbound by SETDB1 and HP1a. Thus, the reason for the targeting of this particular region remains to be explained (Lundberg, 2013).

HP1a has long been known for its repressive function. It was initially identified as a dominant suppressor of position-effect variegation and was named Su(var)205, and it has been reported that HP1a represses gene expression on chromosome 4. However, several studies have reported an activating function of HP1a. The current study suggests that these conflicting reports can at least be partly explained by the observation that HP1a has different functions in different regions; chromosome 4 genes are, on average, repressed, whereas pericentromeric genes are stimulated. It is therefore believed that it is important to look at different groups of genes when studying the effects of HP1a. Otherwise, these opposing effects may cancel each other out on a genome-wide level (Lundberg, 2013).

Nevertheless, the conflicting results cannot be fully explained by the current findings. For example, another study found that transcription was reduced in an RNAi-mediated HP1a knock-down, in contrast to the current results. Therefore, technical differences between experiments should also be considered; in the current study, the possibility of a maternal contribution of HP1a cannot be excluded, as mutants were studied in first-instar larvae from heterozygous mothers, and it is thus likely that we have a reduction in HP1a levels rather than complete removal. It has been shown that maternal HP1a contributes to ~20% of the HP1a protein found in heterozygous mutant first-instar larvae. Previously studies have shown that the average level of gene expression of chromosome 4 is comparable with, or even higher than, that of genes on other chromosomes. At least to some extent, this is a consequence of POF-mediated stimulation of gene expression output, which counteracts the repressing nature of the 4th chromosome (Johansson, 2007; Stenberg, 2009; Johansson, 2012). It is hypothesized that due to POF and other factors, genes on the 4th chromosome have evolved to be functional in this repressive chromatin environment. A decrease in HP1a is mainly expected to cause a reduction of the low affinity binding of HP1a in the gene body, and consequently a de-repression of gene expression. However, prolonged loss or very strong depletion of HP1a will most probably have dramatic effects on the overall structure of chromosome 4 chromatin, and thus lead to a dysfunctional chromatin structure with decreased gene expression. This implies that the genes have adapted to be properly expressed in the local chromatin environment (Lundberg, 2013).

Previous studies have shown that POF is involved in stimulating expression of active genes on chromosome 4. The observed effects in the Pof mutant on genes in the pericentromeric regions and region 2L:31 are most likely explained by indirect effects when HP1a is being redistributed from chromosome 4 to other binding sites, as binding of HP1a to chromosome 4 is dependent on the presence of POF (Riddle, 2012; Figueiredo, 2012; Johansson, 2007). The increased transcriptional output of chromosome 4 genes in the Setdb1 mutant is likely due to loss of the repressive methylation marks, which in turn will reduce HP1a binding. Although it is known that HP1a binding to promoters is independent of methylation marks, it is possible that HP1a binding remains in promoters, where it exerts an activating function (Lundberg, 2013).

The increased chromosome 4 expression observed in the Su(var)3-9 mutant is surprising but could be explained by indirect effects; it is speculated that when Su(var)3-9 is lost from the pericentromeric regions, SETDB1 is redirected from chromosome 4 to sustain normal H3K9 methylation in the pericentromeric regions, thus decreasing HP1a binding to chromosome 4. This could explain why both the Setdb1 and the Su(var)3-9 mutants give such similar up-regulating effects on chromosome 4 expression. An alternative explanation for this effect is that the observed binding of Su(var)3-9 to chromosome 4 has a yet-unknown repressing function independent of the HKMT function of Su(var)3-9 (Lundberg, 2013).

The HP1a Pof double-mutant displayed weak non-significant up-regulation of chromosome 4, with marginally larger error bars than for the HP1a mutant, which supports the suggested balancing mechanism of chromosome 4, where HP1a and POF fine-tune the transcriptional output; in the absence of both components, the overall expression will not change but individual genes will start losing proper transcriptional control (Lundberg, 2013).

Although previous studies have indicated that SETDB1 and Su(var)3-9 have separate main targets, the data show that the majority of genes that are up- or down-regulated in Su(var)3-9 mutants are correspondingly up- or down-regulated in Setdb1 mutants. These results suggest that a redundancy exists between these two proteins, in which both proteins, to some extent, have the ability to be redirected to other locations when needed, as we know that Su(var)3-9 has a chromosome 4 binding capacity. Alternatively, the HP1a system might affect a number of genetic networks so that even if different regions are affected by Su(var)3-9 and Setdb1, the same genetic networks may be indirectly affected. Because Su(var)3-9 affects larger regions than Setdb1, it is likely that more HP1a will be released and redirected to other regions in the Su(var)3-9 mutant than in a Setdb1 mutant, thus causing repression of genes normally unbound by HP1a. This would explain why more genes are down-regulated in the Su(var)3-9 mutant compared with the Setdb1 mutant (Lundberg, 2013).

These results provide strong support for the suggested model in which transposons are repressed by HP1 proteins, as shown for HP1a, the HP1 homolog Rhino and also Su(var)3-9. In contrast, neither SETDB1 nor POF had any effects on transposon expression. Because SETDB1 is known to have a role in repression of chromosome 4, one could speculate that SETDB1 has a greater influence on repression of transposons located specifically on chromosome 4 than in other parts of the genome. However, due to the repetitive nature of the transposons and the methods used in this study, it was not possible to distinguish effects for transposons in specific regions (Lundberg, 2013).

The observation that chromosome 4 displays a stronger effect on non-ubiquitously expressed genes (NUEGs), both in terms of down-regulation in the Pof mutant and up-regulation in the HP1a mutant, is supported by previous findings on chromosome 4. One potential explanation for this is that NUEGs have evolved to respond to a regulatory mechanism, whereas UEGs are more robust in expression. Although weak, it is noteworthy that the effect of the HP1a mutant in the pericentromeric regions (decreased gene expression) was slightly stronger for UEGs than NUEGs; this is in line with the relatively strong binding peak found in promoters compared with the gene body in pericentromeric UEGs, as it has been proposed that HP1a in the promoter has a stimulating effect and HP1a in the gene body of chromosome 4 genes has an inhibiting effect. Note that the number of NUEGs exceeds the number of UEGs on a whole-genome level and on chromosome 4, whereas in pericentromeric regions, the UEGs are over-represented (Lundberg, 2013).

In summary, these data support a model in which HP1a binding to promoters in general has a positive function for transcriptional output, whereas HP1a binding in gene bodies has a negative function. If binding in the gene body is relatively large compared with binding to the promoter, the negative function will dominate. In contrast, if the binding to the promoter is stronger than the gene body, the stimulating effect will be larger, albeit not dominating. A reduction in HP1a levels will initially affect the low-affinity gene binding and sequentially, the loss of HP1a will also affect the promoter binding (Lundberg, 2013).

The average binding level of HP1a is constant irrespective of gene length (the HP1a binding per length unit is constant), implying that longer genes have more HP1a molecules bound in total. This finding, in combination with the suggestion that the repressive effect of HP1a is mainly observed in the gene body, could explain the greater de-repression of longer genes. Stronger binding of HP1a to long genes has also been suggested in previous studies. In addition, some chromatin marks mostly associated with active chromatin have been shown to bind differently to different gene lengths, suggesting that gene length affects the level of association with chromatin marks. Furthermore, there are indications that HP1 proteins are involved with transcription machinery; the mammalian HP1 isoform gamma and H3K9me3 regulate transcriptional activation by associating with the RNA polymerase II (RNP2), and HP1 can interact with and guide the recruitment of the histone chaperone complex FACT to active genes, which facilitates RNP2 transcription elongation. This, along with the current findings, suggests a mechanism in which HP1a is involved in transcriptional elongation. It is speculated that HP1a slows down the progression of the RNP2 through the length of the gene body. HP1a binding mechanisms could also be connected with RNA interactions, as HP1a has been shown to directly interact with RNA transcripts and heterogeneous nuclear ribonucleoproteins, and HP1a association to centric regions in Drosophila and mice is sensitive to RNase treatment (Lundberg, 2013).

Interestingly, it was discovered that a group of non-annotated short genes (<0.5 kb) were repressed by HP1a, even though the HP1a binding levels appeared to be low (which might be explained by technical aspects in determining the binding levels). The lack of annotation and lower wild-type expression level suggest that this group consists of many short genes encoding ncRNAs (Lundberg, 2013).

In the pericentromeric regions of the genome, an interesting connection was observed between the binding and stimulating effects of HP1a and the position of the gene; the closer the gene is located to the centromeric chromatin, the more strongly HP1a binds and stimulates it (Lundberg, 2013).

In conclusion, it was found that HP1a has opposite functions in different genomic regions, repressing expression on chromosome 4 and stimulating expression in pericentromeric regions. Furthermore, the targets of Su(var)3-9 and SETDB1 are considerably more redundant than previously reported, and the overlap between HP1a, Su(var)3-9 and SETDB1 on chromosome 4 genes is extensive. It is however important to note that the different effects caused by HP1a, SETDB1, Su(var)3-9 and POF could all be interrelated to create a balanced genome. Therefore, it is hard to distinguish the separate effects caused by the different proteins (Lundberg, 2013).

Protein Interactions

Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila

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)w^m4h 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).

Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing

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).

The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster

In polytene chromosomes of Drosophila melanogaster, regions of pericentric heterochromatin coalesce to form a compact chromocenter and are highly underreplicated. Focusing on study of X chromosome heterochromatin, it was demonstrated that loss of either SU(VAR)3-9 histone methyltransferase activity or HP1 protein differentially affects the compaction of different pericentric regions. Using a set of inversions breaking X chromosome heterochromatin in the background of the Su(var)3-9 mutations, it was shown that distal heterochromatin (blocks h26-h29) is the only one within the chromocenter to form a big 'puff'-like structure. The 'puffed' heterochromatin has not only unique morphology but also very special protein composition as well: (1) it does not bind proteins specific for active chromatin and should therefore be referred to as a pseudopuff and (2) it strongly associates with heterochromatin-specific proteins SU(VAR)3-7 and SUUR, despite the fact that HP1 and HP2 are depleted particularly from this polytene structure. The pseudopuff completes replication earlier than when it is compacted as heterochromatin, and underreplication of some DNA sequences within the pseudopuff is strongly suppressed. So, it was shown that pericentric heterochromatin is heterogeneous in its requirement for SU(VAR)3-9 with respect to the establishment of the condensed state, time of replication, and DNA polytenization (Demakova, 2007).

In Su(var)2-5 and Su(var)3-7 mutants the chromocenter in salivary gland nuclei looks relatively loose and diffuse (Spierer, 2005). Loss or drastic reduction in the HMTase activity of SU(VAR)3-9 also results in strong decompaction of pericentric heterochromatin (PH) in polytene chromosomes. Most likely, only the functional complex of all these proteins can form compact pericentric heterochromatin. It was of interest to find out to what degree the compact state of heterochromatin is dependent on specific heterochromatin proteins, particularly SU(VAR)3-9. Using a set of chromosome rearrangements placing different parts of X chromosome heterochromatin adjacent to euchromatin, it was discovered that different portions of the polytene Xh are very differently affected by loss of SU(VAR)3-9: only the distal part of heterochromatin (heterochromatic blocks h26-h29 of the metaphase chromosome map becomes decondensed to a varying extent, while morphology of the heterochromatic blocks h30-h34 appears unaffected (Demakova, 2007).

The extent of heterochromatin decompaction depends on several different factors. The pseudopuff is more pronounced in males than in females. Although MSL2 protein, the core component of the dosage compensation complex (DCC), was not found in the pseudopuff, it is suggested that dosage compensation, a process equalizing X-linked gene expression in both sexes, might be responsible for this effect. It is known that DCC is distributed rather discretely, particularly, skipping over intercalary heterochromatin (IH) regions along the male X. Nevertheless, the whole X has a decondensed appearance and underreplication in IH regions is highly suppressed due to DCC function. So, it can be proposed that in males the 'puffed' portion of pericentric Xh relocated into the vicinity of euchromatin becomes dependent on dosage-compensating mechanisms, similarly to IH regions. Another possibility is that the Y chromosome, which represents an additional factor competing for the compaction proteins, might also contribute to the decompaction of Xh and to pseudopuff formation. To note, the fully heterochromatic Y chromosome comprises 40.9 Mb of DNA, while Xh contains ∼20 Mb (Demakova, 2007).

Among the inversions analyzed, w^m4 produces the largest pseudopuff. A good explanation for this effect is currently missing; possibly, the chromatin environment in this eu-heterochromatin junction might contribute to Xh puffing, or some of the DNA sequences might be differentially represented in Xh in different inversions. And finally, decondensation is most strongly expressed on the background of two mutations, Su(var)3-9 and SuUR, despite the fact that SuUR mutation itself does not induce puffing of Xh. The SuUR mutation results in additional polytenization of some of the Xh regions and thus it might increase the amount of Xh material capable of forming the pseudopuff. So, it is believed that loss of both proteins, SUUR and SU(VAR)3-9, has an additive effect on the sizes of the decompacted region (Demakova, 2007).

The region of decondensed heterochromatin that can be called the pseudopuff does not demonstrate signs of true transcriptionally active puffs: the proteins characteristic for active decondensed chromatin (Z4, MSL2, JIL-1, and H3Me3K4) were not detected in the pseudopuff, with the exception of a very weak signal of PolIIo. At the same time, in the Su(var)3-9 mutants, HP1 and HP2 are weakly associated with the region 20F1-4, whereas SUUR and SU(VAR)3-7, in contrast, intensively bind the entire body of the pseudopuff. Recruitment of SU(VAR)3-7 into the pseudopuff in the absence of HP1 and SU(VAR)3-9 appears to be a very specific characteristic of pseudopuff heterochromatin since HP1 and SU(VAR)3-7 proteins cooperate closely in chromosome organization and development (Spierer, 2005). Presence of the SUUR and SU(VAR)3-7 in decompacted chromatin of the pseudopuff might indicate that they do not participate in the process of compaction of this heterochromatic material or that they act in this direction only in the complex with functional SU(VAR)3-9 (Demakova, 2007).

It is interesting to note that different parts of Xh respond differentially to the removal of this complex. The question then, is which features of organization permit proximal heterochromatin to maintain its dense packing in the absence of HP1 and SU(VAR)3-9 activities? It might be proposed that these regions are under control of protein complexes of another composition. For example, these complexes might not utilize HMTase SU(VAR)3-9. However, data on position-effect variegation contradict this idea, since gene inactivation induced by chromosome rearrangements in proximal heterochromatin also depends on the SU(VAR)3-9. It could be suggested that these complexes include some additional compacting proteins. It is known that, in contrast to the distal part of PH, its proximal part is enriched with satellite sequences that are associated with some specific proteins. For, example, the AT-hook protein D1 specifically binds to AT-rich satellites in deep Xh (Demakova, 2007).

In the course of investigating pseudopuff replication it was found that underreplication of the heterochromatic sequences was suppressed in the region of the eu-heterochromatic junction of the w^m4 inversion in Su(var)3-9 mutant. Thus, full polytenization of at least a 45-kb fragment adjacent to euchromatin occurs. The pseudopuff region, in general, completes replication before the end of S-phase; in other words, it does so earlier than the bulk of PH and even some IH regions. Still, a notable fraction of X chromosome heterochromatin sequences remains underreplicated in the polytene chromocenter. It can be assumed that some Xh regions not only do not complete replication but also do not start it. Probably, these regions are separated from replicating chromatin by some barriers that prevent progression of replication forks from adjacent replicons. Therefore, this study demonstrates that Su(var)3-9⁰⁶ may act as a suppressor of underreplication. However, it is not known how this mutation can affect underreplication in other heterochromatic regions (Demakova, 2007).

Replication timing of the pseudopuff is notably changed in the absence of essential changes in transcriptional activity (the PolIIo painting of the pseudopuff looks no more intensive than that of the chromocenter). Moreover, the H3K4 methylation mark characteristic of active regions was not found in the pseudopuff at all. At the same time there exists a correlation between the shift to earlier replication and chromatin decompaction. This observation is interesting in relation to cause-effect relationships among replication timing, transcriptional activity, and decompaction of chromatin (Demakova, 2007).

It is interesting to note that the effect of the SuUR mutation, known as suppression of underreplication, was found not only in PH but also in all IH regions and that these regions complete replication earlier in SuUR mutants than in wild-type ones. The pseudopuff material in SuUR mutants is virtually at the same level of polytenization as in the Su(var)3-9 mutant. However, the SuUR mutation does not involve decompaction of the distal Xh. The same was noted for IH regions. Even if SuUR does induce decompaction of high-level chromatin structures, this is not detected by cytological means. Therefore, SuUR mutation affects replication timing differently compared to Su(var)3-9 (Demakova, 2007).

Summing up, it can be concluded that the reaction of pericentric heterochromatin to loss of SU(VAR)3-9 and HP1 varies in different regions of X heterochromatin. Only the distal part of it undergoes decondensation and forms a new structure called a pseudopuff, which has a specific organization, demonstrating some characteristics of active chromatin: decompaction and, concomitantly, earlier completion of replication. At the same time, the pseudopuff does not contain proteins of active chromatin but does contain several heterochromatic proteins (Demakova, 2007).

The RNA helicase Rm62 cooperates with Su(var)3-9 to re-silence active transcription in Drosophila melanogaster

Gene expression is highly dynamic and many genes show a wide range in expression over several orders of magnitude. This regulation is often mediated by sequence specific transcription factors. In addition, the tight packaging of DNA into chromatin can provide an additional layer of control resulting in a dynamic range of gene expression covering several orders of magnitude. During transcriptional activation, chromatin barriers have to be eliminated to allow an efficient progression of the RNA polymerase. This repressive chromatin structure has to be re-established quickly after it has been activated in order to tightly regulate gene activity. This study shows that the DExD/H box containing RNA helicase Rm62 is targeted to a site of rapid induction of transcription where it is responsible for an increased degree of methylation at H3K9 at the heat shock locus after removal of the heat shock stimulus. The RNA helicase interacts with the well-characterized histone methyltransferase Su(var)3-9 via its N-terminus, which provides a potential mechanism for the targeting of H3K9 methylation to highly regulated genes. The recruitment of Su(var)3-9 through interaction with a RNA helicase to a site of active transcription might be a general mechanism that allows an efficient silencing of highly regulated genes thereby enabling a cell to fine tune its gene activity over a wide range (Boeke, 2011).

This study has identified Rm62 as an interactor with the N-terminus of Su(var)3-9. Interestingly, this interaction domain is shared between the Su(var)3-9 and eIF2γ and could therefore mediate the interaction between Rm62 and both proteins. This study focused on the analysis of the nuclear interaction of Rm62 and Su(var)3-9 as it seems to be important for the efficient shut down of highly activated genes such the hsp70. In accordance with the previously described role of histone modifications at the heat shock locus, a strong H3K9 methylation was observed at the hsp70 gene before heat shock activation that disappears after heat shock and slowly reappears when cells recover from heat shock. This methylation is highly dependent on the presence of Rm62 as it is strongly reduced in Rm62 mutant fly strains. Rm62 mutation not only leads to less H3K9 methylation at the heat shock loci but also leads to a global reduction of the H3K9me2 mark in euchromatin. This suggests a widespread mechanism of methyltransferase recruitment mediated by the interaction between Rm62 and Su(var)3-9 (Boeke, 2011).

Histone modifications play a crucial role in regulating gene expression. The hsp70 locus provides an excellent model promoter for rapidly switching between the on and the off state of transcription and it has been shown to be regulated at multiple levels including histone modification. One of the factors that get recruited to the heat shock promoter immediately after activation is the Rm62, which this study identifies as an interactor with the histone methyltransferase Su(var)3-9. Despite being recruited immediately after heat shock, Rm62 plays a role in transcriptional shut down after removal of the heat shock. It has been suggested that the RNA helicase activity is required for the efficient removal of the RNA from its site of transcription, which in turn is important for the resilencing of the gene. However, a more direct role in the generation of the repressed state could not be excluded. Since a strong, Rm62 dependent recruitment of Su(var)3-9 to the promoter was observed after heat shock that is important for the reestablishment of H3K9 methylated chromatin, it is proposed that the interaction between the two proteins contributes to the regeneration of a repressive chromatin structure after heat shock. Buszczak observed a prolonged phosphorylation of H3S10 at the hsp70 locus in flies that carry a mutation in Rm62 that may very well be due to a failure of recruiting Su(var)3-9 and H3K9 methylation in absence of Rm62. The phosphorylation of H3S10 is severely impaired when the neighboring residue (H3K9) is methylated by Su(var)3-9 in vitro. The recruitment of a H3K9 methyltransferase to the hsp70 gene after heat shock may therefore prevent an efficient phosphorylation of H3S10 thereby favoring the reestablishment of a repressed chromatin structure. At the same time could the increased recruitment of a H3S10 kinase prevent a premature methylation of K9 via the recruited methyltransferases, which may explain the striking kinetic difference observed between the binding of Su(var)3-9 and the accumulation of H3K9 methylated histones. These findings may therefore provide another example of a phospho-methyl switch where a strong interdependence of histone methylation and histone phosphorylation is observed on adjacent residues. Alternatively, the lack of histone methylation after heat shock that is seen by immunofluorescence and by ChIP could be due to the fact that histones are completely removed after heat shock and are only reassembled during recovery. In this case the recruitment of Su(var)3-9 would lead to an increased local concentration of the methyltransferase at the site of the promoter, which could (re-)methylate the ejected histones leading to the regeneration of a repressed state after heat shock. This may in fact also explain the seemingly paradoxical effect of HP1 localisation at heat shock puffs. The binding data could also suggest that the recruitment of Su(var)3-9 is in fact important for gene activation, since it is found to bind to the promoter immediately after the induction of transcription. However, this is unlikely, since an effect of Su(var)3-9 and Rm62 removal is observed on the shut down of hsp70 transcription but not on it's induction (Boeke, 2011).

An alternative explanation for the apparent discrepancy between Su(var)3-9 binding and H3K9 methylation could be the need for an additional signal for the enzyme to become active. Such a signal could be an external signal such as a posttranslational modification or an internal signal such as the RNA transcribed from the hsp70 locus itself. Immediately after heat shock a short burst of small RNAs can be detected that are released from the heat shock locus. Considering the fact that Rm62 also plays a role in RNAi mediated silencing, this pulse of small RNAs might in fact be the cause for the heat shock dependent recruitment of Rm62 to the hsp70 locus that this study observed. The data suggest that Su(var)3-9 is then recruited to the hsp70 locus via protein-protein interactions where it methylates the histones that are assembled onto the promoter during repression. However, the possibility was not tested that the RNA stimulates the activity of Su(var)3-9, which could also contribute to the delayed histone methylation (Boeke, 2011).

Finally it cannot be excluded that, in addition to Su(var)3-9, a demethylase is recruited to the hsp70 locus, which removes the histone methylation from the promoter bound histones. Indeed, the jmjC family member dUTX, which contains a H3K27 specific demthylase associates with the elongating RNA polII enzyme and is recruited to the hsp70 locus after heat shock. It is very likely that multiple redundant mechanisms play a role in the re-silencing of the hsp70 genes after heat shock with all the possibilities discussed above being involved. In light of the novel finding of a functional interaction between Su(var)3-9 and Rm62 it will be interesting to investigate whether this interaction my also provide a mechanistic link between the shut down of highly active genes and the silencing of repetitive DNA elements via the generation of short non translated transcripts that may help in recruiting a histone methyltransferase. Similar mechanisms have been shown to operate in S. pombe but were so far not identified in higher eukaryotes (Boeke, 2011).

A major epigenetic programming mechanism guided by piRNAs

A central enigma in epigenetics is how epigenetic factors are guided to specific genomic sites for their function. It has been reported that a Piwi-piRNA complex associates with piRNA-complementary transposon targets in the Drosophila genome and regulates their epigenetic state. This study reports that Piwi-piRNA complexes bind to numerous piRNA-complementary sequences throughout the genome, implicating piRNAs as a major mechanism that guides Piwi and Piwi-associated epigenetic factors to program the genome. To test this hypothesis, it was demonstrated that inserting piRNA-complementary sequences to an ectopic site leads to Piwi, HP1a, and Su(var)3-9 recruitment to the site as well as H3K9me2/3 enrichment and reduced RNA polymerase II association, indicating that piRNA is both necessary and sufficient to recruit Piwi and epigenetic factors to specific genomic sites. Piwi deficiency drastically changed the epigenetic landscape and polymerase II profile throughout the genome, revealing the Piwi-piRNA mechanism as a major epigenetic programming mechanism in Drosophila (Huang, 2013).

This study has systematically demonstrated the existence of the Piwi-piRNA epigenetic guidance mechanism and its function as a major mechanism of guiding epigenetic factors to their target sites in Drosophila. This mechanism provides a clear and effective answer to the long-standing question on how epigenetic factors are recruited to their specific target sites to achieve epigenetic programming throughout the genome. Given that some other Piwi proteins and piRNAs also exist in the nucleus of other organisms including mammals, this mechanism might have profound significance in diverse organisms (Huang, 2013).

Whole-genome mapping produced the high-resolution map of Piwi and piRNA binding to the genome. The perfect colocalization of Piwi and piRNA binding sites is expected given their association as molecular complexes. These Piwi-piRNA complexes directly bind to many regions in the genome, exerting epigenetic repression at most of the target sites. This may account for the diverse biological functions of Piwi in different cell types during development. In particular, many Piwi-piRNA complexes bind to transposon sequences; this may be a major mechanism that is responsible for transposon silencing by Piwi as reported in many studies (Huang, 2013).

Furthermore, this analysis has revealed, at the whole-genome scale, the dependence of HP1a and H3K9 methylation on Piwi, which suggests that HP1a and histone methylases are recruited by Piwi-piRNA complexes as a major mechanism to many sites in the genome. It is important to note that Lei and colleagues reported that, in several piRNA clusters, HP1 binding is apparently unaffected by Argonaute proteins, including Piwi (Moshkovich and Lei, 2010). This result was fully anticipated because Piwi colocalizes with HP1a at many, but not all, HP1a-containing bands on polytene chromosomes. The binding of HP1a to chromatin at Piwi-free sites must be via a different mechanism, possibly via the canonical H3K9me2/3- mediated mechanism. These data, combined with the current findings, indicate that there are at least two different ways for recruiting HP1a to the chromatin, with Piwi-piRNA mechanism as a main way of recruitment, as demonstrated in this study and suggested by previous polytene staining data (Huang, 2013).

The results demonstrate that piRNA is both necessary and sufficient to bring Piwi to specific genomic sites in a sequence-specific manner and reveal a crucial role of piRNA in guiding epigenetic factors to specific sites in the genome. This epigenetic guidance mechanism is similar to the RNAi-mediated heterochromatin formation in the fission yeast in that both are mediated by small RNAs and Piwi/Ago. However, it distinctly differs from the yeast pathway in three major aspects. First, it recruits HP1a without H3K9 methylation, which then leads to recruitment of HMT and H3K9 methylation. This is in stark contrast to the yeast RNAi pathway in which the RITS complex first recruits HMT, which then leads to the methylation of H3K9 and eventual recruitment of HP1. In addition, this is also in sharp contrast to the known H3K9 methylation-dependent mechanism of HP1a recruitment in higher eukaryotes and represents a novel H3K9 methylation-independent mechanism. The recruitment of HMT by HP1a would lead to H3K9 methylation, which would result in further recruitment of HP1a molecules to the site, thereby stabilizing the repressive state of the chromatin. Second, the Piwi-piRNA-mediated epigenetic guidance mechanism can lead to transcriptional repression or activation, depending on the genomic context Last, the Piwi-piRNA mechanism involves single-stranded piRNAs and Piwi proteins rather than double- stranded siRNAs and Ago proteins. Given the genomic complexity of the higher eukaryotes, piRNAs, mostly 24-32 nt in length, are ideal candidate molecules for conferring sequence specificity in a genome-wide context. Indeed, the extreme complexity of the identified piRNAs, with more than 20,000 piRNAs associated with Piwi alone in Drosophila and more than 58,000 piRNAs in mammals, renders the Piwi-piRNA pathway a likely major epigenetic factor guidance mechanism in Drosophila, and possibly even in mammals (Huang, 2013).

High-resolution mapping analysis suggests that piRNAs might associate with euchromatin by binding to nascent RNA transcripts of 100-800 bp yet with heterochromatin by directly binding to DNA. This is in perfect agreement with previous observation that Piwi binding to euchromatin and heterochromatin is sensitive to RNaseIII and RNaseH that selectively digest double-stranded RNA and RNA-DNA hybrid, respectively. Given the complexity of the heterochromatic context, it is not clear so far exactly how piRNA binds to heterochromatic DNA. However, it is conceivable that such direct binding might occur between piRNA and single-stranded DNA (e.g., during DNA replication or transcription) or between piRNA and DNA duplex. Future studies will resolve these hypotheses (Huang, 2013).

It is also worthy noting that the sequence specificity of piRNA binding to its targets is additive with respect to individual base pairs. Each mismatch compromises the piRNA binding efficacy by 40%, so that a piRNA carrying three point mutations retains only
10% its binding ability to target sequences. This is in contrast to siRNA targeting that requires perfect complementarity and miRNA targeting that requires a mismatch in the middle position yet requires perfect match in the base 2-7 'seed sequences'. Indeed, the graded sequence specificity of piRNA binding to its target sequences might create a mechanism of quantitative regulation that allows piRNAs to guide Piwi and epigenetic factors to even more genomic sites with graded effects as well as bestows tolerance to point mutations that frequently occur in heterochromatic and repetitive sequences (Huang, 2013).

The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila

RNA surveillance factors are involved in heterochromatin regulation in yeast and plants, but less is known about the possible roles of ribonucleases in the heterochromatin of animal cells. This study shows that RRP6, one of the catalytic subunits of the exosome, is necessary for silencing heterochromatic repeats in the genome of Drosophila melanogaster. It was shown that a fraction of RRP6 is associated with heterochromatin, and the analysis of the RRP6 interaction network reveals physical links between RRP6 and the heterochromatin factors HP1a, SU(VAR)3-9 and RPD3. Moreover, genome-wide studies of RRP6 occupancy in cells depleted of SU(VAR)3-9 demonstrates that SU(VAR)3-9 contributes to the tethering of RRP6 to a subset of heterochromatic loci. Depletion of the exosome ribonucleases RRP6 and DIS3 stabilizes heterochromatic transcripts derived from transposons and repetitive sequences, and renders the heterochromatin less compact, as shown by micrococcal nuclease and proximity-ligation assays. Such depletion also increases the amount of HP1a bound to heterochromatic transcripts. Taken together, these results suggest that SU(VAR)3-9 targets RRP6 to a subset of heterochromatic loci where RRP6 degrades chromatin-associated non-coding RNAs in a process that is necessary to maintain the packaging of the heterochromatin (Eberle, 2015).

Approximately 30% of the genome of Drosophila melanogaster is heterochromatic and is made up of transposons, transposon fragments and repetitive sequences with different degrees of complexity. The heterochromatin contains high levels of heterochromatin-specific proteins, such as Heterochromatin Protein 1a (HP1a), and is enriched in certain patterns of post-translational modifications of the histone tails. Heterochromatin formation involves a cascade of histone modifications that are targeted to specific regions of the genome by complex protein-protein and protein-nucleic acid interactions. In the switch from euchromatin to heterochromatin, acetylated H3K9 (H3K9ac) is deacetylated by histone deacetylases such as RPD3/HDAC1. H3K9 is subsequently methylated by histone methyltransferases, and the methylated H3K9 (H3K9me) acts as a binding site for HP1a. The properties of the heterochromatin can spread along the chromatin fiber, and HP1a plays a central role in this process. The ability of HP1a to dimerize, to interact with the methyltransferase SU(VAR)3-9, and to bind H3K9me provides the basis for the spreading of heterochromatin. An additional level of complexity in the establishment of heterochromatic states is provided by the fact that HP1a can also bind RNA in both D. melanogaster and Schizosaccharomyces pombe. Recent studies on Swi6, the HP1a ortholog of S. pombe, have shown that the interaction of Swi6 with RNA interferes with the binding of Swi6 with H3K9me (Eberle, 2015).

Small non-coding RNAs are essential components of the regulation of chromatin packaging in different organisms. Fission yeast uses siRNAs to silence heterochromatic sequences through the recruitment of the H3K9 methyltransferase Clr4. RNAi-dependent mechanisms of heterochromatin assembly exist also in plants, where siRNAs direct de novo DNA methyltransferases to specific genomic sequences. Animal cells use instead the piRNA pathway to trigger heterochromatin assembly and transposon silencing in the germ line. In D. melanogaster, non-coding RNAs transcribed from transposon-rich regions are processed into piRNAs, and a 'Piwi-piRNA guidance hypothesis' has been recently proposed for the recruitment of SU(VAR)3-9 and HP1a to heterochromatin. The Piwi-piRNA system is active during early development and it directs the initial establishment of heterochromatin states not only in the germ line but also in somatic cells. Recent studies suggest that after embryogenesis, the patterns of heterochromatin packaging are maintained through cell divisions via piRNA-independent mechanisms (Eberle, 2015).

An important player in the regulation of non-coding RNAs is the exosome, a multiprotein complex with ribonucleolytic activity. In D. melanogaster, the core of the exosome associates with two catalytic active subunits, RRP6 and DIS3. In the cell nucleus, the exosome is involved in the processing of many non-coding RNAs, including pre-rRNAs, and in the quality control of mRNA biogenesis. The exosome ribonucleases also degrade a large variety of unstable, non-coding RNAs in various organisms including S. cerevisiae, plants, and animals. Moreover, recent studies have revealed that RRP6 participates in the regulation of enhancer RNAs and in the degradation of unstable transcripts synthesized at DNA double-strand breaks (Eberle, 2015).

The exosome has been functionally linked to the methylation of H3K9 in heterochromatin. In S. pombe, RRP6 participates in the assembly of centromeric heterochromatin through an RNAi-independent mechanism, and collaborates with the RNAi machinery to silence developmentally regulated loci and retrotransposons. Much less is known about the possible links between RRP6 and heterochromatin in animals. This study found that a fraction of RRP6 is associated with heterochromatin in the genome of D. melanogaster, and physical interactions has been identifiedf between RRP6 and several heterochromatin factors, including HP1a, SU(VAR)3-9, and RPD3. These results show that SU(VAR)3-9 promotes the targeting of RRP6 to transposon-rich heterochromatic loci. In these loci, RRP6 contributes to maintaining the structure of the heterochromatin by degrading non-coding RNAs that would otherwise compromise the packaging of the chromatin (Eberle, 2015).

This study shows that RRP6 interacts physically with HP1a and SU(VAR)3-9, and that RRP6 is associated with a subset of heterochromatic regions of the genome. Less RRP6 is bound to the heterochromatin in cells with reduced levels of SU(VAR)3-9, which indicates that SU(VAR)3-9 contributes to the targeting of RRP6 to heterochromatin. Although the RNAi experiments do not reveal whether the effect of SU(VAR)3-9 knockdown on RRP6 occupancy is direct or indirect, the fact that RRP6 and SU(VAR)3-9 colocalize and can be co-immunoprecipitated suggests that SU(VAR)3-9 facilitates the recruitment of RRP6 to the heterochromatin, or stabilizes the interaction of RRP6 with other chromatin components, through a physical interaction (Eberle, 2015).

This study has focused on RRP6, and the existence of multiple exosome subcomplexes in cells of D. melanogaster makes it difficult to establish whether the entire exosome has a role in the heterochromatin. However, two observations suggest that this is the case. Firstly, the simultaneous depletion of both catalytic subunits of the exosome, RRP6 and DIS3, gave additive effects on the levels of chromatin-associated RNAs and on the association of HP1a to heterochromatic RNAs. Secondly, it was previously shown that a fraction of RRP4, a core exosome subunit, is also associated with chromatin. Altogether, these observations suggest that the entire exosome, not RRP6 alone, is targeted to heterochromatic loci through an interaction with SU(VAR)3-9 (Eberle, 2015).

Depletion of RRP6 or simultaneous depletion of RRP6 and DIS3 led to a local increase in heterochromatic transcripts associated with subtelomeric and pericentromeric regions, without a significant increase in the density of RNA Pol-II at those regions. This suggests that under normal conditions the RRP6 and DIS3 degrade pervasive RNAs that are transcribed from the heterochromatin. Direct MNase assays and PLA-based assays designed to measure the compaction of the chromatin revealed that the depletion of the exosome ribonucleases loosens the structure of the heterochromatin in the regions that accumulate heterochromatic non-coding RNAs, without affecting the levels of H3K9 methylation or the association of SU(VAR)3-9 with the chromatin. In S. pombe, deletion of the rrp6 gene leads to a derepression of heterochromatin, and this effect is partly due to the fact that in the absence of RRP6 activity, aberrant RNA species accumulate in S. pombe and recruit the siRNA machinery in competition with the RNAi-dependent pathways of H3K9 methylation. The situation is different in D. melanogaster, as no change in H3K9me2 or SU(VAR)3-9 recruitment occurred when RRP6 and DIS3 were depleted (Eberle, 2015).

What is then the mechanism by which the exosome ribonucleases influence the compaction of the heterochromatin in D. melanogaster? The HP1a ortholog in S. pombe, Swi6, is an RNA-binding protein, and non-coding RNAs can cause the eviction of Swi6 from the S. pombe heterochromatin by competing with H3K9me for Swi6. The HP1a protein of D. melanogaster interacts with several RNA-binding proteins and can bind directly to RNA. This study has shown that depletion of RRP6 and DIS3 results in increased levels of non-coding transcripts associated with heterochromatin in D. melanogaster cells. HP1a-RIP signals at selected heterochromatic loci are also increased in cells depleted of RRP6 and DIS3. Altogether, these observations are consistent with a model in which RRP6, and perhaps also DIS3, participate in the degradation of heterochromatic non-coding RNAs that, if stabilized, would outcompete the binding of HP1a to the methylated H3K9 and would thereby disrupt the packaging of the heterochromatin (Eberle, 2015).

Heterochromatin domains are characterized by high levels of H3K9me2 and by the presence of HP1a and SU(VAR)3-9. These results show that RRP6 interacts with SU(VAR)3-9 and that this interaction is important to tether RRP6 to the heterochromatin. Transcripts derived from sporadic transcription of heterochromatic repeat sequences are kept at low levels by RRP6 degradation. Failure to degrade such transcripts results in increased levels of chromatin-associated transcripts, increased binding of HP1 to the chromatin-associated transcripts, and chromatin decondensation (Eberle, 2015).

Specialized protein-protein interactions target RRP6 to different chromatin environments RRP6 and the exosome act on many different types of transcripts and participate in many essential biological processes. The existence of multiple mechanisms to target RRP6 to different types of transcripts, or even to different nuclear compartments, is thus not unexpected. The association of the exosome-or exosome subunits- with genes transcribed by RNA polymerase II (Pol-II) is mediated by interactions with different types of proteins. Co-immunoprecipitation experiments in D. melanogaster identified SPT5 and SPT6, two transcription elongation factors, as interaction partners for the exosome, which led to the proposal that the exosome is tethered to the transcription machinery during transcription elongation. In D. melanogaster, the exosome is also tethered to protein-coding loci through interactions with the hnRNP protein HRP59/RUMP. In human cells, a NEXT complex containing MTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7 mediates an interaction between the exosome and Pol-II transcripts through the nuclear cap-binding complex. In many cases, these intermolecular interactions target the exosome to genomic loci that produce relatively stable transcripts, for instance protein-coding transcripts or stable non-coding RNAs. In these loci, the role of the exosome is primarily linked to RNA surveillance, not turnover (Eberle, 2015).

Much less is known about the mechanisms that target the exosome or its individual subunits to non-protein coding RNAs in the heterochromatin. This study of the RRP6 interactome in cells of D. melanogaster has revealed interactions between RRP6 and heterochromatin factors, and has established an important role for SU(VAR)3-9 in determining RRP6 occupancy. Depletion of SU(VAR)3-9 has a profound effect on the association of RRP6 with a subset of chromatin regions, including many transposon loci. The present findings suggest that these regions, that can be referred to as 'SUV-dependent', produce transcripts that are actively degraded by RRP6. SU(VAR)3-9 has less impact on the targeting of RRP6 to euchromatic protein-coding genes, where interactions with the Pol-II machinery and with mRNA-binding proteins play instead a decisive role. Altogether, the picture that emerges from many studies is that specialized protein-protein interactions target RRP6 to specific genomic environments where RRP6 participates in the processing, surveillance or degradation of specific RNA substrates (Eberle, 2015).

DEVELOPMENTAL BIOLOGY

The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila

A new class of small RNAs (endo-siRNAs) produced from endogenous double-stranded RNA (dsRNA) precursors was recently shown to mediate transposable element (TE) silencing in the Drosophila soma. These endo-siRNAs might play a role in heterochromatin formation. This has been shown in S. pombe for siRNAs derived from repetitive sequences in chromosome pericentromeres. To address this possibility, the viral suppressors of RNA silencing B2 and P19 were used. These proteins normally counteract the RNAi host defense by blocking the biogenesis or activity of virus-derived siRNAs. It was hypothesized that both proteins would similarly block endo-siRNA processing or function, thereby revealing the contribution of endo-siRNA to heterochromatin formation. Accordingly, P19 as well as a nuclear form of P19 expressed in Drosophila somatic cells were found to sequester TE-derived siRNAs whereas B2 predominantly bound their longer precursors. Strikingly, B2 or the nuclear form of P19, but not P19, suppressed silencing of heterochromatin gene markers in adult flies, and altered histone H3-K9 methylation as well as chromosomal distribution of histone methyl transferase Su(var)3-9 and Heterochromatin Protein 1 in larvae. Similar effects were observed in dcr2, r2d2, and ago2 mutants. These findings provide evidence that a nuclear pool of TE-derived endo-siRNAs is involved in heterochromatin formation in somatic tissues in Drosophila (Fagegaltier, 2009).

This study implicates components of the RNAi pathway in heterochromatin silencing during late Drosophila development. The study also provides correlative evidence supporting a functional link between endo-siRNAs and the formation or maintenance of somatic heterochromatin in flies. The viral proteins NLS-P19 and B2 suppress the silencing of PEV markers and induce aberrant distribution of H3m2K9 and H3m3K9 heterochromatic marks as well as histone H3 methylase Su(var)3-9 in larval tissues. Dcr2 and Ago2 mutations have similar effects. In striking contrast, cytoplasmic P19 has no noticeable effect on chromatin. It is proposed that B2 inhibits Dcr2-mediated processing of double-stranded TE read-through transcripts in the cytoplasm; it is further proposed that NLS-P19 sequesters TE-derived siRNA duplexes. This model implies that part of the cytoplasmic pool of TE-derived endo-siRNA (which might be involved in PTGS events) is translocated back into the nucleus to exert chromatin-based functions. In C. elegans, silencing of nuclear-localized transcripts involves nuclear transport of siRNAs by an NRDE-3 Argonaute protein. A similar siRNA nuclear translocation system, possibly mediated by Ago2, may also exist in flies. Alternatively, an as yet unidentified siRNA duplex transporter may be involved. Deep sequencing analyses show that the fraction of siRNAs sequestered by NLS-P19 is smaller as compared with the one bound by P19 in the cytoplasm. Thus, the poor effects of P19 on nuclear gene silencing may be explained if the cytoplasmic pool of siRNA competes with the pool of siRNA to be translocated in the nucleus (Fagegaltier, 2009).

The Dcr-1 partner Loquacious (Loqs), but not the Dcr-2 partner R2D2, was unexpectedly found to be required for biogenesis of siRNA derived from fold-back genes that form dsRNA hairpins. By contrast, it is noteworthy that loqs mutations had little or no impact on the accumulation of siRNA derived from TE. The finding that r2d2 but not loqs mutation suppresses the silencing of PEV reporters and delocalizes H3m2K9 and H3m3K9 heterochromatic marks agrees with these results and further suggests that siRNA involved in heterochromatin formation and siRNA derived from endogenous hairpins arise from distinct r2d2- and loqs-dependent pathways, respectively. One possible mechanism by which TE- or repeat-derived endo-siRNAs could promote heterochromatin formation is by tethering complementary nascent TE transcripts and guiding Su(var)3-9 recruitment and H3K9 methylation. Identifying which enzymes tether siRNAs to chromatin in animals is a future challenge. In addition, some endo-siRNAs could also impact on heterochromatin formation by posttranscriptionaly regulating the expression of chromatin modifiers, such as Su(var)3-9. In any case, the current results demonstrate the value of viral silencing suppressor proteins in linking siRNAs to heterochromatin silencing in the fly soma, as established in S. pombe and higher plants. Because silencing suppressors are at the core of the viral counterdefensive arsenal against antiviral RNA silencing in fly, whether they also induce epigenetic changes in chromatin states during natural infections by viruses deserves further investigation (Fagegaltier, 2009).

Effects of Mutation or Deletion

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).

Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila

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, pitkin^D, 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-1^Su(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-1^Su(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)3-9^ptn 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)3-9^ptn 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)3-9^ptn 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)3-9^ptn 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-1^Su(var)3-1 alleles display a strong Su(var) effect. Therefore JIL-1^Su(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-1^Su(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-1^Su(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-1^Su(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-1^Su(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)3-9^ptn 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-1^Su(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-1^Su(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-1^Su(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).

Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing

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).

The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila

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-1^Su(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-1^Su(var)3-1 proteins are mislocalized to ectopic chromosome sites. Thus, it is proposed that the dominant gain-of-function effect of the JIL-1^Su(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-1^Su(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).

High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants

The structural and functional analyses of heterochromatin are essential to understanding how heterochromatic genes are regulated and how centromeric chromatin is formed. Because the repetitive nature of heterochromatin hampers its genome analysis, new approaches need to be developed. This study describes how, in double mutants for Su(var)3-9 and Suppressor of Under-Replication (SuUR) genes, encoding two structural proteins of heterochromatin, new banded heterochromatic segments appear in all polytene chromosomes due to the strong suppression of under-replication in pericentric regions. FISH on salivary gland polytene chromosomes from these double mutant larvae allows high resolution of heterochromatin mapping. In addition, immunostaining experiments with a set of antibodies against euchromatic and heterochromatic proteins reveal their unusual combinations in the newly appeared segments: binding patterns for HP1 and HP2 are coincident, but both are distinct from H3diMetK9 and H4triMetK20. In several regions, partial overlapping staining is observed for the proteins characteristic of active chromatin RNA Pol II, H3triMetK4, Z4, and JIL1, the boundary protein BEAF, and the heterochromatin-enriched proteins HP1, HP2, and SU(VAR)3-7. The exact cytological position of the centromere of chromosome 3 was visualized on salivary gland polytene chromosomes by using the centromeric dodeca satellite and the centromeric protein CID. This region is enriched in H3diMetK9 and H4triMetK20 but is devoid of other proteins analyzed (Andreyeva, 2007).

dSETDB1 and SU(VAR)3–9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster

Germline-stem cells (GSCs) produce gametes and are thus true 'immortal stem cells'. In Drosophila ovaries, GSCs divide asymmetrically to produce daughter GSCs and cystoblasts, and the latter differentiate into germline cysts. This study shows that the histone-lysine methyltransferase dSETDB1, located in pericentric heterochromatin, catalyzes H3-K9 trimethylation in GSCs and their immediate descendants. As germline cysts differentiate into egg chambers, the dSETDB1 function is gradually taken over by another H3-K9-specific methyltransferase, SU(VAR)3–9. Loss-of-function mutations in dsetdb1 (eggless) or Su(var)3–9 abolish both H3K9me3 and heterochromatin protein-1 (HP1) signals from the anterior germarium and the developing egg chambers, respectively, and cause localization of H3K9me3 away from DNA-dense regions in most posterior germarium cells. These results indicate that dSETDB1 and SU(VAR)3–9 act together with distinct roles during oogenesis, with dsetdb1 being of particular importance due to its GSC-specific function and more severe mutant phenotype (Yoon, 2008).

This study shows that dSETDB1 is the only HKMTase responsible for the synthesis of H3K9me3 signals in the inner germarium where GSCs and their early descendants are found. When these vasa-positive cells move to region-3 germarium, the H3-K9 trimethylating task is transferred to a combination of dSETDB1 and SU(VAR)3–9, as both enzymes act cooperatively in all other somatic-type cells of the germarium. After the egg chamber buds off from the germarium, the trimethylation activity is now entirely the province of SU(VAR)3–9. The results disclose that the developmental program uses dSETDB1 first and then SU(VAR)3–9 during GSC differentiation, indicating that the two HKMTases perform distinct functions in these germ cells. The role of dSETDB1 in early GSC differentiation is presumably to 'pre-mark' certain regions of chromatin, including the pericentric heterochromatin, with H3K9me3. The biochemical features of these pre-marked regions might be different from those of regions that are substrates of SU(VAR)3–9, and the pre-marked regions may be the platform on which incoming SU(VAR)3–9 further modulates the pre-methylated chromatin regions in later-developing VASA-positive cells. The functional significance of trimethylating, or 'priming', GSC chromatins with dSETDB1 is highlighted by the catastrophic ovarian phenotypes observed in, and the sterility of, the dsetdb1 female homozygote. By contrast, although the egg chambers of the Su(var)3–9¹⁷ mutant completely lacked H3K9me3 signals, which might be expected to result in a phenotype more severe than that of the dsetdb1 mutant, the Su(var)3–9¹⁷ mutant is capable of oogenesis, is able to lay eggs, and is fertile (Yoon, 2008).

Meanwhile, the localization of both dSETDB1 and Su(var)3-9 at DAPI-dense heterochromatin does not necessarily mean that they target the same chromatin loci in early- and late-stage of oogenesis, respectively. The observation that dSETDB1, but not Su(VAR)3-9, is essential for Drosophila oogenesis provides a possibility that the two HKMTases may have different sets of target chromatin regions during oogenesis. It would be interesting to examine whether dsetdb1 phenotypes could be rescued or not if exogenous Su(var)3-9 were expressed at high level in GSCs and their close derivatives (Yoon, 2008).

In germ cells, dSETDB1 locates at DAPI-dense, pericentric heterochromatin. This was unexpected because the mammalian counterpart, SETDB1/Eset, is known to have euchromatin-associated function. Seum (2007) recently reported that, in Drosophila polytene chromosomes, dSETDB1 locates at the fourth chromosome. This fourth chromosome is known to be unusual as it has many characteristics of heterochromatic domains (such as a high-repeat density, no recombination and late replicating) and, at the same time, it shows features of euchromatin (such as being transcriptionally active and having a high gene density); in fact, many of the genes in the fourth chromosome are expressed during development. These characteristics indicate that the banded regions of the fourth chromosome are different from pericentric heterochromatin, which highlights the peculiarity of dSETDB1 localization to DAPI-dense heterochromatin in the germarium (Yoon, 2008).

The location of dSETDB1 at pericentric heterochromatin probably indicates that dSETDB1 participates, at a global level, in regulating chromosome organization and maintaining the chromosome integrity in the germ-lineages. This hypothesis is supported by the observation that the main H3K9me3 spots were displaced and went astray from DNA-dense heterochromatin regions in most region-3 cells of the dsetdb1^G19561 germarium. In addition, the egg chambers of the dsetdb1^G19561 mutant ovary that survived stage six were shown to have disorganized chromosomes in the nurse cell nuclei. The nurse cell chromosomes of the stage-7 egg chambers in both wild-type and Su(var)3–9¹⁷ ovaries were organized into bundles with well-developed, large nucleoli, whereas those in the dsetdb1^G19561 ovaries were simply scattered throughout the nucleoplasm without nucleolar regions; otherwise, all were stained positive in the TUNEL assay. HP1 was diffusely located in these nuclei of dsetdb1^G19561 mutant egg chambers but the HP1 mislocalization is unlikely to be the reason for the scattered chromosomes because the Su(var)3–9¹⁷ egg chambers totally lacked HP1 but had nucleolar regions between bundles of chromosomes. These results suggest that dSETDB1 has a role in coordinating the chromosomal integrity in the germ-cell lineages, and the loss of dSETDB1 function results in a dysregulation of chromosome organization (Yoon, 2008).

In the ovary, the main type of methylation catalyzed by dSETDB1 is H3K9me3. In the dsetdb1^G19561 germarium, the loss of H3K9me3 was limited to the germ cells in the inner germarium. By contrast, in the salivary glands, dSETDB1 primarily synthesizes H3K9me2 at the fourth chromosome at which dSETDB1 itself localizes. Alterations in the H3K9me3 pattern and intensity were not detected in the polytene chromosomes in these studies. This means that dsetdb1 synthesizes either H3K9me2 or H3K9me3, depending on the type of cells in which it functions. By analogy with mammalian SETDB1/Eset, dSETDB1 can produce in vitro all the methylation types such as H3K9me1, H3K9me2 and H3K9me3. Under in vivo conditions, the specificity of SETDB1 activity and the resulting state of methylation depend on regulatory protein(s) associated with SETDB1/Eset. This is shown by the observation that a murine ATFa-associated factor (mAM) tightly associates with SETDB1 and facilitates the SETDB1-dependent conversion of H3K9me2 to H3K9me3. Therefore, the proteins that regulate SETDB1 activity determine the H3-K9 methylation state in certain tissue cells and at particular developmental stages, and this might be true for dSETDB1 in Drosophila (Yoon, 2008).

Relating to likely dSETDB1-associated protein(s), a clue was provided by the observation that a dsetdb1 null mutant, DmSetdb1^10.1a, dies at the late pupal stage, but it can be rescued to progress to the adult stage by expression of a truncated DmSETDB1^421–1,261 transgene, which was constructed by deleting the N-terminal 420 amino acids of the full-length dSETDB1. Of particular interest is the finding that the rescued females are sterile whereas the males are fertile, which is the same phenotype as seen with the dsetdb1^G19561 mutant. This rescue experiment indicates that the truncated DmSETDB1^421–1,261 is enough for the null DmSetdb1^10.1a mutants to survive the pupal stage, but is still insufficient to overcome the female sterility. This provides important clues about the tissue and substrate specificity of dSETDB1. This indicates that the N-terminal region (spanning 1–420 amino acids) of dSETDB1 is instrumental in female fertility. This region likely forms a functional domain that provides a binding site(s) for regulatory protein(s) that positions dSETDB1 at pericentric heterochromatin in the PGCs and GSC-derived cells instead of the fourth chromosomes, and preferentially synthesizes H3K9me3 instead of H3K9me2. Mammalian SETDB1/ESET is known to associate with several transcriptional regulators such as the ERG protein, mAM, KRAB-zinc-finger protein KAP1, and MBD1/MCAF1. It would be interesting to investigate the factor(s) that restricts dSETDB1 to the germ-cell lineages and favors H3K9me3 over H3K9me2 in the ovary (Yoon, 2008).

At present, there is no information on SU(VAR)3-9 function during the Drosophila oogenesis. Because an antibody capable of immunocytochemically detecting SU(VAR)3-9 protein was unavailable, a transgenic line was used that expresses GFP-tagged SU(VAR)3-9 protein as an alternative. It is clear that the ectopic expression pattern shown by the GFP-tagged SU(VAR)3-9 does not always reflect the pattern of endogenous SU(VAR)3-9. Nevertheless, if the SU(VAR)3-9-eGFP were expressed in a cell with endogenous SU(VAR)3-9, the SU(VAR)3-9-eGFP signals would be localized to wherever endogenous SU(VAR)3-9 is located. The results of RISH and RT-PCR analyses showed that Su(var)3-9 is expressed in the ovarioles including the germarium and participates in oogenesis, and the egg chambers were shown to lack H3K9me3 in the Su(var)3–9¹⁷ mutant flies. Therefore, a GFP-tagged SU(VAR)3-9 transgenic fly was used to determine the location of endogenous SU(VAR)3-9 from the ectopically expressed SU(VAR)3-9-eGFP signals in the ovarian cells, and the results showed that the SU(VAR)3-9-eGFP signals were overlapped with H3K9me3/HP1 signals in the egg chambers, indicating that endogenous SU(VAR)3-9 is responsible for H3K9me3 signals in developing egg chambers. The function of SU(VAR)3-9 in the germarium could also be deduced from the localization of SU(VAR)3-9-eGFP signals. In the inner germarium SU(VAR)3-9-eGFP signals were less co-localized with H3K9me3 signals than in the outer germarium. Such a positioning of SU(VAR)3-9-eGFP in the germarium is in agreement with the prediction of endogenous SU(VAR)3-9 function in the outer germarium. Therefore, it is certain that SU(VAR)3-9 also has a role in the oogenesis. Despite its role as an influential epigenetic modifier, the SU(VAR)3-9 function during the oogenesis is likely to be dispensable because Su(var)3-9 null mutant flies are fertile (Yoon, 2008).

HP1 recognizes H3K9me2 and H3K9me3. In the polytene chromosomes of salivary glands, HP1 localizes at the chromocenter and chromosome 4, in agreement with the pattern of H3K9me2, rather than H3K9me3, which is present at the core of the chromocenter. Mutations in the dsetdb1 gene abolish both H3K9me2 and HP1 signals from the fourth chromosome in the salivary glands. By contrast, the HP1 in the nuclei of both the germarium and the developing egg chambers mainly associates with H3K9me3 instead of H3K9me2. The dsetdb1^G19561 germarium and the Su(var)3–9¹⁷ egg chambers have normal-looking H3K9me2 signals but lack H3K9me3, and their nuclei also lack HP1 signals. These observations indicate that in some cells and tissues, HP1 binds either H3K9me2 or H3K9me3, and the preferred substrate depends on the HKMTase(s) itself that recruits and tethers HP1 to their sites of action (Yoon, 2008).

In summary, this study has demonstrated that dsetdb1 is expressed, in a germ cell-specific manner, in the germarium; the germline stem cells and their early descendants reside in the anterior part of the germarium and both H3K9me3 and HP1 signals are abolished with mutations in the dsetdb1 gene. In the GSC-derived cells, dSETDB1 trimethylates H3-K9 residues at pericentric heterochromatin, but this function is performed by SU(VAR)3–9 as germline cysts differentiate into egg chambers. Loss-of-function mutation in Su(var)3–9 abolishes both H3K9me3 and HP1 signals in developing egg chambers. Both dSETDB1 and SU(VAR)3-9 collaborate in the region-3 germarium and a mutation in either of these genes causes localization of H3K9me3 away from DNA-dense regions in the region-3 cells. These findings, therefore, indicate that dsetdb1 and Su(var)3-9 act sequentially to regulate chromosome organization in accordance with the differentiation of the germline-stem cells in Drosophila (Yoon, 2008).

A balance between euchromatic (JIL-1) and heterochromatic [SU(var)2-5 and SU(var)3-9] factors regulates position-effect variegation in Drosophila

This study shows that the haplo-enhancer effect of JIL-1 has the ability to counterbalance the haplo-suppressor effect of both Su(var)3-9 and Su(var)2-5 on position-effect variegation, providing evidence that a finely tuned balance between the levels of JIL-1 and the major heterochromatin components contributes to the regulation of gene expression (Wang, 2011).

The essential JIL-1 histone H3S10 kinase is a major regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, the major heterochromatin markers H3K9me2, HP1a [Su(var)2-5], and Su(var)3-7 spread to ectopic locations on the chromosome arms. These observations suggested a model for a dynamic balance between euchromatin and heterochromatin, where, as can be monitored in position-effect variegation (PEV) arrangements, the boundary between these two states is determined by antagonistic functions of a euchromatic regulator (JIL-1) and the major determinants of heterochromatin assembly, e.g., Su(var)3-9, HP1a, and Su(var)3-7. In support of this model, it has been shown that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on gene expression using PEV assays (Deng, 2010); however, potential dynamic interactions between JIL-1 and the other two heterochromatin genes, Su(var)3-9 and Su(var)2-5, were not addressed previously. Interestingly, in other genetic interaction assays monitoring the lethality as well as the chromosome morphology defects associated with the null JIL-1 phenotype, only a reduction in the dose of the Su(var)3-9 gene rescued both phenotypes. In contrast, in the same assays a reduction of Su(var)3-7 rescued the lethality, but not the chromosome defects (Deng, 2010), and no genetic interactions were detectable between JIL-1 and Su(var)2-5. Thus, these findings indicate that while Su(var)3-9 activity may be a major factor in the lethality and chromatin-structure perturbations associated with loss of the JIL-1 histone H3S10 kinase, these effects are likely to be uncoupled from HP1a and, to a lesser degree, from Su(var)3-7. This raises the question of whether JIL-1 dynamically interacts with the two other heterochromatin genes, Su(var)2-5 and Su(var)3-9, in regulating gene expression, as it does with Su(var)3-7 (Wang, 2011).

To answer this question, the effect of various combinations of loss-of-function alleles of JIL-1 and Su(var)3-9 or Su(var)2-5 was explored on PEV caused by the P-element insertion line 118E-10. Insertion of this P element (P[hsp26-pt, hsp70-w]) into euchromatic sites results in a uniform red-eye phenotype whereas insertion into a known heterochromatin region of the fourth chromosome results in a variegating eye phenotype. It has been demonstrated that loss-of-function JIL-1 alleles can act as haplo-enhancers of PEV, resulting in increased silencing of gene expression (Deng, 2010), whereas loci for structural components of heterochromatin such as Su(var)3-9, Su(var)2-5, and Su(var)3-7 act as strong haplo-suppressors. In the experiments, the transgenic reporter line 118E-10 was crossed into JIL-1^z2/+, Su(var)3-9⁰⁶/+, and Su(var)2-5⁰⁵/+ mutant backgrounds as well as into JIL-1^z2/Su(var)3-9⁰⁶ and JIL-1^z2/Su(var)2-5⁰⁵ double-mutant backgrounds. The JIL-1^z2 allele is a true null allele, the loss-of-function Su(var)3-9⁰⁶ allele is due to a DNA insertion, and the Su(var)2-5⁰⁵ loss-of-function allele is associated with a frameshift resulting in a nonsense peptide containing only the first 10 amino acids of HP1a. Thus, to test whether the heterozygous JIL-1^z2 allele could counterbalance the suppression of the Su(var)3-9⁰⁶ or Su(var)2-5⁰⁵ loss-of-function alleles of the PEV of 118E-10, the eye pigment levels of the various genotypes were compared. Pigment assays were performed using three sets of 10 pooled fly heads from each genotype. Although both male and female flies were scored, due to sex differences only results from male flies are shown. However, the trend observed in female flies was identical to that in male flies. The heterozygous JIL-1^z2/+ genotype enhances PEV as indicated by the increased proportion of white ommatidia and a 59% decrease in the optical density (OD) of the eye pigment levels. This reduction was statistically significant. In contrast, the heterozygous Su(var)3-9⁰⁶/+ and Su(var)2-5⁰⁵/+ genotypes suppress PEV as indicated by an increase of the proportion of red ommatidia and a statistically significant increase, respectively, in the OD of the eye pigment levels. However, in the JIL-1^z2/Su(var)3-9⁰⁶ and JIL-1^z2/Su(var)2-5⁰⁵ double-mutant backgrounds, variegation of the proportion of red ommatidia was intermediate, and the eye pigment levels were statistically indistinguishable from genotypes with +/+ levels of JIL-1, Su(var)3-9, and Su(var)2-5 proteins (Wang, 2011).

To test whether a heterozygous JIL-1 null allele also could counterbalance the suppression of the Su(var)3-9⁰⁶ or Su(var)2-5⁰⁵ alleles of the PEV of w^m4, experiments were performed similar to those described above for 118E-10. For male flies, the heterozygous JIL-1^z2/+ genotype enhances PEV as indicated by a 67% decrease in the OD of the eye pigment levels. This reduction was statistically significant. In contrast, the heterozygous Su(var)3-9⁰⁶/+ and Su(var)2-5⁰⁵/+ genotypes suppress PEV . However, in the JIL-1^z2/Su(var)3-9⁰⁶ and JIL-1^z2/Su(var)2-5⁰⁵ double-mutant backgrounds, the eye pigment levels were significantly reduced by 13% and 17% as compared to heterozygous Su(var)3-9⁰⁶/+ and Su(var)2-5⁰⁵/+ genotypes, indicating that a heterozygous JIL-1 null allele has the ability to counterbalance the suppression of the Su(var)3-9⁰⁶ or Su(var)2-5⁰⁵ alleles of the PEV of w^m4. However, it should be noted that it has been demonstrated that JIL-1 can act both as an enhancer and as a suppressor of w^m4 PEV, depending on the precise levels of JIL-1. Thus, the genetic interactions between JIL-1 and the Su(var)3-9 and Su(var)2-5 alleles in regulating the PEV of w^m4 are likely to be more complex than in the case of 118E-10 where reduced levels of JIL-1 always act as an enhancer. In females where the enhancer effect of the heterozygous JIL-1^z2 allele is less pronounced than in males, a statistically significant counterbalancing effect was detected only in flies of the JIL-1^z2/Su(var)2-5⁰⁵ genotype (Wang, 2011).

These results demonstrate that the haplo-enhancer effect of JIL-1 has the ability to counterbalance the haplo-suppressor effect of both Su(var)3-9 and Su(var)2-5 on the PEV of two different alleles. In previous experiments, a genetic interaction between JIL-1 and Su(var)2-5 was not detected. However, the assays used to probe for interactions were viability and rescue of polytene chromosome morphology. As indicated by the experiments presented in this study, these parameters are likely to be independent of and separate from the mechanisms contributing to epigenetic regulation of PEV and gene silencing. Consequently, the present experiments, taken together with those of Deng, (2010) using a JIL-1 null allele, provide strong evidence that a finely tuned balance between the levels of JIL-1 and all of the major heterochromatin components Su(var)3-9, HP1a, and Su(var)3-7 contributes to the regulation of PEV and gene expression (Wang, 2011).

Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark

The JIL-1 kinase mainly localizes to euchromatic interband regions of polytene chromosomes and is the kinase responsible for histone H3S10 phosphorylation at interphase in Drosophila. However, recent findings raised the possibility that the binding of some H3S10ph antibodies may be occluded by the H3K9me2 mark obscuring some H3S10 phosphorylation sites. Therefore, this study has characterized an antibody to the epigenetic H3S10phK9me2 double mark as well as three commercially available H3S10ph antibodies. The results showed that for some H3S10ph antibodies their labeling indeed can be occluded by the concomitant presence of the H3K9me2 mark. Furthermore, it was demonstrated that the double H3S10phK9me2 mark is present in pericentric heterochromatin as well as on the fourth chromosome of wild-type polytene chromosomes but not in preparations from JIL-1 or Su(var)3-9 null larvae. Su(var)3-9 is a methyltransferase mediating H3K9 dimethylation. Furthermore, the H3S10phK9me2 labeling overlapped with that of the non-occluded H3S10ph antibodies as well as with H3K9me2 antibody labeling. Interestingly, when a Lac-I-Su(var)3-9 transgene is overexpressed, it upregulates H3K9me2 dimethylation on the chromosome arms creating extensive ectopic H3S10phK9me2 marks suggesting that the H3K9 dimethylation occurred at euchromatic H3S10ph sites. This is further supported by the finding that under these conditions euchromatic H3S10ph labeling by the occluded antibodies was abolished. Thus, these findings indicate a novel role for the JIL-1 kinase in epigenetic regulation of heterochromatin in the context of the chromocenter and fourth chromosome by creating a composite H3S10phK9me2 mark together with the Su(var)3-9 methyltransferase (Wang, 2014).

REFERENCES

Aagaard, L., et al. (1999). Functional mammalian homologues of the Drosophila PEV-modifier Su(var)3-9 encode centromere-associated proteins which complex with the heterochromatin component M31. EMBO J. 18: 1923-1938. 10202156

Aagaard, L., Schmid, M., Warburton, P. and Jenuwein, T. (2000). Mitotic phosphorylation of SUV39H1, a novel component of active centromeres, coincides with transient accumulation at mammalian centromeres. J. Cell Sci. 113: 817-29. 10671371

Ahmed, S., Saini, S., Arora, S. and Singh, J. (2001). Chromodomain protein Swi6-mediated role of DNA polymerase alpha in establishment of silencing in fission yeast. J. Biol. Chem. 276(51): 47814-21. 11581276

Alder, O., et al. (2010). Ring1B and Suv39h1 delineate distinct chromatin states at bivalent genes during early mouse lineage commitment. Development 137(15): 2483-92. PubMed Citation: 20573702

Allshire, R. C., et al. (1995). Mutations derepressing silent centromeric domains in fission yeast disrupt chromosome segregation. Genes Dev. 9(2): 218-33. 7851795

Andreyeva, E. N., et al. (2007). High-resolution analysis of Drosophila heterochromatin organization using SuUR Su(var)3-9 double mutants. Proc. Natl. Acad. Sci. 104(31): 12819-24. PubMed Citation: 17640911

Andrulis, E. D., Werner, J., Nazarian, A., Erdjument-Bromage, H., Tempst, P. and Lis, J. T. (2002). The RNA processing exosome is linked to elongating RNA polymerase II in Drosophila. Nature 420: 837-841. PubMed ID: 12490954

Bannister, A. J., et al. (2001). Selective recognition of methylated lysine 9 on histone H3 by the HP1 chromo domain. Nature 410: 120-124. 11242054

Boeke, J., Bag, I., Ramaiah, M. J., Vetter, I., Kremmer, E., Pal-Bhadra, M., Bhadra, U. and Imhof, A. (2011). The RNA helicase Rm62 cooperates with Su(var)3-9 to re-silence active transcription in Drosophila melanogaster. PLoS One 6: e20761. PubMed ID: 21674064

Czermin, B., et al. (2001). Physical and functional association of SU(VAR)3-9 and HDAC1 in Drosophila. EMBO Reports 2: 915-919. 11571273

Czvitkovich, S., et al. (2001). Over-expression of the SUV39H1 histone methyltransferase induces altered proliferation and differentiation in transgenic mice. Mech. Dev. 107(1-2): 141-53. 11520670

Danzer, J. R. and Wallrath, L. L. (2004). Mechanisms of HP1-mediated gene silencing in Drosophila. Development 131: 3571-3580. 15215206

Demakova, O. V., et al. (2007). The SU(VAR)3-9/HP1 complex differentially regulates the compaction state and degree of underreplication of X chromosome pericentric heterochromatin in Drosophila melanogaster. Genetics 175(2): 609-20. PubMed Citation: 17151257

Deng, H., et al. (2010). JIL-1 and Su(var)3-7 interact genetically and counteract each other's effect on position-effect variegation in Drosophila. Genetics 185: 1183-1192. PubMed Citation: 20457875

Dialynas, G. K., Makatsori, D., Kourmouli, N., Theodoropoulos, P. A., McLean, K., Terjung, S., Singh, P. B. and Georgatos, S. D. (2006). Methylation-independent binding to histone H3 and cell cycle-dependent incorporation of HP1beta into heterochromatin. J Biol Chem 281: 14350-14360. PubMed ID: 16547356

Di Stefano, L., Ji, J. Y., Moon, N. S., Herr, A. and Dyson, N. (2007). Mutation of Drosophila Lsd1 disrupts H3-K4 methylation, resulting in tissue-specific defects during development. Curr. Biol. 17(9): 808-12. Medline abstract: 17462898

Eberle, A.B., Jordán-Pla, A., Gañez-Zapater, A., Hessle, V., Silberberg, G., von Euler, A., Silverstein, R.A. and Visa, N. (2015). An interaction between RRP6 and SU(VAR)3-9 targets RRP6 to heterochromatin and contributes to heterochromatin maintenance in Drosophila melanogaster. PLoS Genet 11: e1005523. PubMed ID: 26389589

Ebert, A., Schotta, G., Lein, S., Kubicek, S., Krauss, V., Jenuwein, T. and Reuter, G. (2004). Su(var) genes regulate the balance between euchromatin and heterochromatin in Drosophila. Genes Dev. 18(23): 2973-83. 15574598

Ekwall, K. and Ruusala, T. (1994). Mutations in rik1, clr2, clr3 and clr4 genes asymmetrically derepress the silent mating-type loci in fission yeast. Genetics 136(1): 53-64. 8138176

Ekwall, K., et al. (1996). Mutations in the fission yeast silencing factors clr4+ and rik1+ disrupt the localisation of the chromo domain protein Swi6p and impair centromere function. J. Cell Sci. 109: 2637-48. 8937982

Eskeland, R.., Czermin, B., Boeke, J., Bonaldi, T., Regula, J.T. and Imhof, A. (2004). The N-terminus of Drosophila SU(VAR)3-9 mediates dimerization and regulates its methyltransferase activity. Biochemistry 43: 3740-3749. 15035645

Fagegaltier, D., et al. (2009). The endogenous siRNA pathway is involved in heterochromatin formation in Drosophila. Proc. Natl. Acad. Sci. 106(50): 21258-63. PubMed Citation: 19948966

Ferres-Marco, D., et al. (2006). Epigenetic silencers and Notch collaborate to promote malignant tumours by Rb silencing. Nature 439(7075): 430-6. 16437107

Figueiredo, M. L., Philip, P., Stenberg, P. and Larsson, J. (2012). HP1a recruitment to promoters is independent of H3K9 methylation in Drosophila melanogaster. PLoS Genet 8: e1003061. PubMed ID: 23166515

Filion, G. J., van Bemmel, J. G., Braunschweig, U., Talhout, W., Kind, J., Ward, L. D., Brugman, W., de Castro, I. J., Kerkhoven, R. M., Bussemaker, H. J. and van Steensel, B. (2010). Systematic protein location mapping reveals five principal chromatin types in Drosophila cells. Cell 143: 212-224. PubMed ID: 20888037

Firestein, R., et al. (2000). Set domain-dependent regulation of transcriptional silencing and growth control by SUV39H1, a mammalian ortholog of Drosophila Su(var)3-9. Mol. Cell. Biol. 20(13): 4900-9. 10848615

Garcia-Cao, M., O'Sullivan, R., Peters, A. H., Jenuwein, T. and Blasco, M. A. (2004). Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases. Nat. Genet. 36: 94-99. 14702045

Greil, F., et al. (2003). Distinct HP1 and Su(var)3-9 complexes bind to sets of developmentally coexpressed genes depending on chromosomal location. Genes Dev. 17: 2825-2838. 14630943

Horita, D. A., et al. (2001). Solution structure, domain features, and structural implications of mutants of the chromo domain from the fission yeast histone methyltransferase Clr4. J. Mol. Biol. 307(3): 861-70. 11273706

Huang, X. A., Yin, H., Sweeney, S., Raha, D., Snyder, M. and Lin, H. (2013). A major epigenetic programming mechanism guided by piRNAs. Dev Cell 24: 502-516. PubMed ID: 23434410

Johnson, W. L., Yewdell, W. T., Bell, J. C., McNulty, S. M., Duda, Z., O'Neill, R. J., Sullivan, B. A. and Straight, A. F. (2017). RNA-dependent stabilization of SUV39H1 at constitutive heterochromatin. Elife 6. PubMed ID: 28760200

Kharchenko, P. V., (2011). Comprehensive analysis of the chromatin landscape in Drosophila melanogaster. Nature 471: 480-485. PubMed ID: 21179089

Krauss, V. and Reuter, G. (2000). Two genes become one: The genes encoding heterochromatin protein SU(VAR)3-9 and translation initiation factor subunit eIF-2gamma are joined to a dicistronic unit in holometabolic insects. Genetics 156: 1157-1167. 11063691

Krauss, V., et al. (2006). The evolution of the histone methyltransferase gene Su(var)3-9 in metazoans includes a fusion with and a re-fission from a functionally unrelated gene. BMC Evol. Biol. 6: 18. 16512904

Ivanova, A. V., et al. (1998). The chromo and SET domains of the Clr4 protein are essential for silencing in fission yeast. Nat. Genet. 19(2): 192-5. 9620780

Lachner, M., et al. (2001). Methylation of histone H3 lysine 9 creates a binding site for HP1 proteins. Nature 410(6824): 116-20. 11242053

Lehnertz, B., et al. (2003). Suv39h-mediated histone H3 Lysine 9 methylation directs DNA methylation to major satellite repeats at pericentric heterochromatin. Curr. Biol. 13: 1192-1200. 12867029

Liu, D. X., et al. (2005). Regulation of neuron survival and death by p130 and associated chromatin modifiers. Genes Dev. 19: 719-732. 15769944

Loyola, A., et al. (2001). Reconstitution of recombinant chromatin establishes a requirement for histone-tail modifications during chromatin assembly and transcription. Genes Dev. 15: 2837-2851. 11691835

Lundberg, L. E., Stenberg, P. and Larsson, J. (2013). HP1a, Su(var)3-9, SETDB1 and POF stimulate or repress gene expression depending on genomic position, gene length and expression pattern in Drosophila melanogaster. Nucleic Acids Res 41: 4481-4494. PubMed ID: 23476027

Melcher, M., et al. (2000). Structure-function analysis of SUV39H1 reveals a dominant role in heterochromatin organization, chromosome segregation, and mitotic progression. Mol. Cell. Biol. 20(10): 3728-41. 10779362

Nakayama, J., et al. (2001). Role of histone H3 lysine 9 methylation in epigenetic control of heterochromatin assembly. Science 292(5514): 110-3. 11283354

Ner, S. S., Harrington, M. J. and Grigliatti, T. A. (2002). A role for the Drosophila SU(VAR)3-9 protein in chromatin organization at the histone gene cluster and in suppression of position-effect variegation. Genetics 162: 1763-1774. 12524347

Nielsen, S. J., et al. (2001). Rb targets histone H3 methylation and HP1 to promoters. Nature 412: 561-565. 11484059

Nishioka, K., et al. (2002). Set9, a novel histone H3 methyltransferase that facilitates transcription by precluding histone tail modifications required for heterochromatin formation. Genes Dev. 16: 479-489. 11850410

O'Carroll, D., et al. (2000). Isolation and characterization of Suv39h2, a second histone H3 methyltransferase gene that displays testis-specific expression. Mol. Cell Biol. 20(24): 9423-33. 11094092

Peters, A. H., et al. (2001). Loss of the Suv39h histone methyltransferases impairs mammalian heterochromatin and genome stability. Cell 107(3): 323-37. 11701123

Rea, S., et al. (2000). Regulation of chromatin structure by site-specific histone H3 methyltransferases. Nature 406(6796): 593-9. 10949293

Rudolph, T., et al. (2007). Heterochromatin formation in Drosophila is initiated through active removal of H3K4 methylation by the LSD1 homolog SU(VAR)3-3. Mol. Cell 26(1): 103-15. Medline abstract: 17434130

Schotta, G., et al. (2002). Central role of Drosophila SU(VAR)3-9 in histone H3-K9 methylation and heterochromatic gene silencing. EMBO J. 21: 1121-1131. 11867540

Shi, S., et al. (2006). JAK signaling globally counteracts heterochromatic gene silencing. Nat. Genet. 38(9): 1071-6. Medline abstract: 16892059

Tschiersch, B., et al. (1994). The protein encoded by the Drosophila position-effect variegation suppressor gene Su(var)3-9 combines domains of antagonistic regulators of homeotic gene complexes. EMBO J. 13(16): 3822-3831. 7915232

Vaquero, A., et al. (2007). SIRT1 regulates the histone methyl-transferase SUV39H1 during heterochromatin formation. Nature 450(7168): 440-4. PubMed citation: 18004385

Wang, C., Girton, J., Johansen, J. and Johansen, K. M. (2011). A balance between euchromatic (JIL-1) and heterochromatic [SU(var)2-5 and SU(var)3-9] factors regulates position-effect variegation in Drosophila. Genetics 188(3): 745-8. PubMed Citation: 21515582

Wang, C., Li, Y., Cai, W., Bao, X., Girton, J., Johansen, J. and Johansen, K. M. (2014). Histone H3S10 phosphorylation by the JIL-1 kinase in pericentric heterochromatin and on the fourth chromosome creates a composite H3S10phK9me2 epigenetic mark. Chromosoma [Epub ahead of print]. PubMed ID: 24429699

Yoon, J, et al. (2008). dSETDB1 and SU(VAR)3–9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster. PLoS ONE 3: e2234. PubMed Citation: 18493619

Zhang, W., Deng, H., Bao, X., Lerach, S., Girton, J., Johansen, J. and Johansen, K. M. (2006). The JIL-1 histone H3S10 kinase regulates dimethyl H3K9 modifications and heterochromatic spreading in Drosophila. Development 133: 229-235. 16339185

Zhang, Y., et al. (2008). Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos. Dev. Cell 14(4): 481-93. PubMed Citation: 18410726

date revised: 30 October 2015

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.