InteractiveFly: GeneBrief

Suppressor of variegation 3-7: Biological Overview | References

In Drosophila, dosage compensation augments X chromosome-linked transcription in males relative to females. This process is achieved by the Dosage Compensation Complex (DCC), which associates specifically with the male X chromosome. It has been found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. This study examined the impact of change in levels of SU(VAR)3-7 on dosage compensation. It was first demonstrated that the DCC makes the X chromosome a preferential target for heterochromatic markers. In addition, reduced or increased amounts of SU(VAR)3-7 result in redistribution of the DCC proteins MSL1 and MSL2, and of Histone 4 acetylation of lysine 16, indicating that a wild-type dose of SU(VAR)3-7 is required for X-restricted DCC targeting. SU(VAR)3-7 is also involved in the dosage compensated expression of the X-linked white gene. Finally, it was shown that absence of maternally provided SU(VAR)3-7 renders dosage compensation toxic in males, and that global amounts of heterochromatin affect viability of ectopic MSL2-expressing females. Taken together, these results bring to light a link between heterochromatin and dosage compensation (Spierer, 2008).

Drosophila uses two systems of whole chromosome regulation: dosage compensation mediating the two fold up-regulation of male X-linked genes and the Painting of fourth POF, regulating the mainly heterochromatic fourth chromosome. Binding of POF to the fourth chromosome is dependent on the heterochromatic protein HP1 (Johansson, 2007). POF and HP1 colocalize on fourth chromosome-linked genes and both are involved in the global regulation of the fourth chromosome. Johansson (2007) proposed that POF stimulates and HP1 represses genes expression and that the interdependent binding of these two proteins precisely tunes output from the fourth chromosome (Spierer, 2008).

Dosage compensation targets the male X chromosome to correct the unbalance between the unique X chromosome of males and the two X chromosomes of females. To compensate for the resulting disparity in X chromosome-linked gene expression, most X-linked genes in males are hyperactivated. The Dosage Compensation Complex (DCC) consists of five proteins called the MSLs for Male Specific Lethal (MSL1, MSL2, MSL3, MLE and MOF) as well as two non-coding RNAs, roX1 and roX2. In males, the expression of MSL2 mediates the stabilization of the other proteins and the assembly of the DCC specifically on the X chromosome. This results in an enrichment of acetylation of histone H4 at lysine 16 (H4K16ac) on the male X chromosome, due to the MOF protein of the complex. The histone mark could in part explain the subsequent hypertranscription of X-linked genes in males. In females, the Sex-lethal gene turns off the dosage compensation system by repressing the Msl2 translation (Spierer, 2008).

One of the most intriguing issues of dosage compensation is the specific recognition of the male X chromosome by the DCC. Searches for X chromosomal DNA sequences determining DCC binding failed to identify a consensus sequence. Global mapping of MSL proteins on the X chromosome has demonstrated that the DCC associates primarily with genes rather than intergenic regions, displays a 3'- bias and correlates with transcription. Moreover, the MSL complex is attracted to genes marked by H3K36 trimethylation, a mark of active transcription. Furthermore, the levels of DCC proteins MSL1 and MSL2 are critical for correct targeting to the X chromosome. Over-expression of both msl1 and msl2 results in inappropriate MSLs binding to the chromocenter and chromosome 4. MSL2, deleted of its C-terminal part, binds as a complex with MSL1 to the heterochromatic chromocenter. roX RNAs are also key components for X chromosome targeting since roX1roX2 mutants cause relocation of MSLs complex to autosomal regions and the chromocenter. These data reveal an unpredicted physical association between the MSL complex and heterochromatic regions (Spierer, 2008 and references therein).

H4K16 acetylation is not the only chromatin mark distinguishing the Drosophila male X chromosome from the autosomes. Phosphorylation of H3 at serine 10, catalyzed by JIL-1, is a histone modification highly enriched on the male X chromosome. The JIL-1 kinase interacts with the DCC and is involved in dosage compensation of X-linked genes. Interestingly, Jil-1 mutant alleles affect both condensation of the male X chromosome and expansion of heterochromatic domains, providing evidence for a dynamic balance between heterochromatin and euchromatin. Other general modulators of chromatin state, as ISWI or NURF, are also required for normal X chromosome morphology in males. The NURF complex and MSL proteins have opposite effects on X chromosome morphology and on roX transcription (Spierer, 2008 and references therein).

An intriguing genetic interaction has been discovered between the heterochromatic proteins SU(VAR)3-7 and HP1, and dosage compensation (Spierer, 2005). Su(var)3-7 encodes a protein mainly associated with pericentromeric heterochromatin and telomeres, but also with a few euchromatic sites (Reuter, 1990; Cléard, 1997; Delattre, 2000). Specific binding to pericentric heterochromatin requires the heterochromatic protein HP1 (Spierer, 2005). HP1 localizes to heterochromatin through an interaction with methylated K9 of histone H3 (H3K9me2), a heterochromatic mark mainly generated by the histone methyltransferase SU(VAR)3-9. SU(VAR)3-7 interacts genetically and physically with HP1 (Cléard, 1997; Delattre, 2000) and with SU(VAR)3-9 (Schotta, 2002; Delattre, 2004). Su(var)3-7, Su(var)205, encoding HP1, and Su(var)3-9 are modifiers of position effect variegation (PEV), the phenomenon of gene silencing induced by heterochromatin. These three genes enhance or suppress the PEV depending on their doses and thus are considered as encoding structural components of heterochromatin. Strikingly, the amounts of SU(VAR)3-7 and HP1 affect male X chromosome morphology more dramatically than other chromosomes. Reduced doses of SU(VAR)3-7 or HP1 result in bloating of the X chromosome specifically in males (Spierer, 2005). Increased doses of SU(VAR)3-7 cause the opposite phenotype, a spectacular condensation of the X chromosome associated with recruitment of other heterochromatin markers (Delattre, 2004). Some unique feature of the male X chromosome makes it particularly sensitive to change in SU(VAR)3-7 amounts. In addition, knock-down of Su(var)3-7 results in reduced male viability leading to a 0.7 male/female ratio in the progeny of Su(var)3-7 homozygous mutant mothers (Delattre, 2004). The possibility of interaction between activating and repressive chromatin factors on the male X chromosome led to an analysis of the impact of SU(VAR)3-7 on dosage compensation (Spierer, 2008).

This study shows that wild-type levels of SU(VAR)3-7 are required for male X chromosome morphology, X chromosome-restricted DCC targeting, expression of P(white) transgenes in males and for coping with increased MSL1 and MSL2 levels. Evidence is provided for interplay between heterochromatin and dosage compensation in Drosophila (Spierer, 2008).

This work reveals a connection between heterochromatin and dosage compensation in Drosophila. SU(VAR)3-7 is implicated in male X chromosome morphology, in correct distribution of the DCC, in the expression of the dosage compensated white gene and in male viability. This study describes some of the complex interactions between SU(VAR)3-7 and the DCC and illustrates the ability of heterochromatin/DCC balance to affecting chromatin conformation and protein distribution. The results support a model whereby the activating dosage compensation system in Drosophila is influenced by chromatin silencing factors (Spierer, 2008).

Reduced levels of SU(VAR)3-7 induce bloating of the male X chromosome, whereas increased levels cause condensation of the male X chromosome (Spierer, 2005; Delattre, 2004). Moreover, at high dose, SU(VAR)3-7, normally restricted to heterochromatin, invades preferentially the male X chromosome and, to a lesser extent, the autosomes (Delattre, 2004). These observations led to an investigation of the features rendering the male X chromosome particularly sensitive to SU(VAR)3-7. This paper examined the genetic interaction between a gene essential for dosage compensation, mle, and Su(var)3-7 on the morphology of the male X chromosome. Bloating and shrinking of the X chromosome both require the presence of the DCC, and assembly of the DCC in females is sufficient to make their X chromosomes preferential targets for SU(VAR)3-7, when in excess. The dosage compensation system is thus responsible for the sensitivity of the male X chromosome to changes in SU(VAR)3-7 amounts. One explanation for the X chromosome sensitivity is that increased levels of H4K16 acetylation induced by the DCC render chromatin of the male X chromosome more accessible to chromatin factors and more sensitive to disturbances than other chromosomes. The possibility cannot be excluded that SU(VAR)3-7-induced X chromosome defects are indicators of a more general effect of the protein on all chromosomes as described for ISWI: Null mutations in the gene encoding ISWI cause aberrant morphology of the male X chromosome but not of autosomes and females X chromosomes, but expression of a very strong dominant negative form of ISWI in vivo leads indeed to decondensation of all chromosomes in both sexes. Nevertheless other data in this study, to be described below, favour the hypothesis whereby X chromosome defects result from a specific interaction between SU(VAR)3-7 and dosage compensation (Spierer, 2008).

Male X chromosome sensitivity to SU(VAR)3-7 was rather unexpected, as in a wild-type context, in contrast to over-expression conditions, no preferential binding of SU(VAR)3-7 to the male X chromosome was detected. The absence of detectable SU(VAR)3-7 enrichment on the male X polytene chromosome from third instar larvae may be due either to lack of sensitivity of the immunostaining procedure or to observations made in inappropriate tissues or development stages. Similar puzzling observations have been made for HP1, which is not preferentially seen on the male X polytene chromosomes, although reduced HP1 induces bloating of the male X chromosome. In cultured cells however, a moderate HP1 enrichment was detected with the DamID technique on the male X chromosome and not on the female X chromosomes, suggesting that HP1 participates in the structure of the male X chromosome (Spierer, 2008).

A striking and novel result of this study is that precise wild-type amounts of the heterochromatic protein SU(VAR)3-7 are required to restrict MSLs binding to the X chromosome. In Su(var)3-7 mutants, it was observed that the MSL proteins are recruited to the chromocenter. Furthermore, when SU(VAR)3-7 is present in excess, MSLs are massively delocalized from the X chromosome to many sites on autosomes (Spierer, 2008).

Two hypotheses are proposed. First, the effect of SU(VAR)3-7 on the MSLs distribution is indirect and due to the regulation of the expression of a component of the DCC. Indeed, increased amounts of MSL1 and MSL2 lead to MSLs binding on autosomes and at chromocenter, and MSLs delocalization from the X chromosome to autosomes and chromocenter is detectable in roX1roX double mutants. A careful regulation of MSLs and roX RNAs concentration is therefore important to restrict DCC activity to appropriate targets. In addition, increased levels of MSL2, or of both MSL2 and MSL1, result in a diffuse morphology of the X chromosome. This phenotype resembles the bloated X chromosome of Su(var)3-7 and Su(var)2-5 mutants, suggesting that the amounts of MSL2 and MSL1 are downregulated by the heterochromatic proteins. Expression of many euchromatic genes are under the control of the HP1 protein, leading the idea of testing whether changes in SU(VAR)3-7 amounts modify the expression of roXs, msl1 and msl2 or the stability of MSL1 and MSL2. Quantitative RTPCR and Western blots did not detect significant changes in the amounts of DCC components. In HP1 mutant msl1 transcription is also not affected. These results speak against the hypothesis of regulation of expression of a DCC component by a SU(VAR)3-7/HP1 complex (Spierer, 2008).

The second hypothesis is that SU(VAR)3-7 modifies the MSLs distribution by changing the chromatin state of the X chromosome and of the pericentric heterochromatin. Changes in chromatin conformation or epigenetic marks could modify affinity of the DCC for entry sites. Numerous entry sites on the X chromosome have been described, and a hierarchy of entry sites has been suggested with different affinities for the DCC. Even cryptic binding sites on autosomes and at the chromocenter are recognized by the DCC in certain conditions. It is proposed that increasing SU(VAR)3-7 amounts on the X chromosome results in an enrichment of HP1 and H3K9 dimethylation, and leads to a more compact heterochromatic-like structure of the X chromosome which then blocks access to the high-affinity entry sites. The free DCC, chased from the X chromosome sites turns toward low-affinity sites present on autosomes, but not toward those embedded into the chromocenter. Indeed, cryptic chromocenter sites become more inaccessible by heterochromatin compaction, a phenomenon also responsible for the enhancement of variegation by increased SU(VAR)3-7 levels (Delattre, 2004). Inversely, the absence of SU(VAR)3-7 induces a more relaxed chromatin state at the chromocenter (Spierer, 2005), thus increasing affinity of the entry sites embedded into heterochromatin, and allowing MSLs binding at the chromocenter. Similar recruitments of MSLs at heterochromatin have been described in the literature in three situations: (1) in roX1roX2 mutants, (2) in presence of excess of MSL2 and in (3) C-terminal truncated MSL2 mutants. This means that cryptic entry sites present in heterochromatin become more accessible to the MSLs either in a Su(var)3-7 mutants or if DCC composition is modified. The explanation of heterochromatin affinity for the MSLs remains obscure. On the X chromosome, the Su(var)3-7 mutation induces the bloated morphology resembling that described as a result of decreased levels of silencing factors as HP1, ISWI and NURF, or of increased MSLs levels. The current study and others suggest that male X chromosome morphology depends on the balance between silencing and activating complexes. The simultaneous existence of the repressive SU(VAR)3-7/HP1 proteins and the MSLs complex may provide a set of potential interactions that cumulatively regulate dosage compensation (Spierer, 2008).

Several arguments support a role for SU(VAR)3-7 in dosage compensation. Reduced male viability in the progeny of Su(var)3-7 homozygous females is a first argument for a function played by the protein specifically in males (Seum, 2002). The results also show that wild-type amounts of SU(VAR)3-7 are required to cope with increased MSL1 and MSL2 levels. In absence of maternal SU(VAR)3-7 product, the transgenes expressing MSL1 and MSL2 become toxic to males, whereas no lethality is observed with wild-type or half amounts of SU(VAR)3-7. This suggests that SU(VAR)3-7 is required very early in development to counteract an excess of MSL1 or MSL2 activity. Corroborating this effect, it was determined that the global amount of heterochromatin affects the viability of females engineered to expressing msl2. The presence of the highly heterochromatic Y chromosome kills half of the females expressing msl2. It has been proposed that the Y chromosome functions as a sink for heterochromatic factors as SU(VAR)3-7 and HP1 (Weiler, 1995; Beckstead, 2005). A Y chromosome added to XX females could sequester heterochromatic proteins, and induce lethality in a context of female dosage compensation. All these data lead to the conclusion that SU(VAR)3-7 is required for the viability of dosage-compensated flies. Two explanations are proposed: 1) Either SU(VAR)3-7 is required to restrict DCC on the X chromosome and the lethality induced by the lack of SU(VAR)3-7 is due to the MSLs ectopic activity outside of the X chromosome (at heterochromatin), or 2) SU(VAR)3-7 is required on the dosage compensated X chromosome and, in this case, the Su(var)3-7 mutant lethality results from malfunctioning of the DCC on the X (Spierer, 2008).

To discriminate between these two hypothesis, expression of X-linked genes was examined in Su(var)3-7 mutants. Although small changes are visible, the RT-PCR analysis did not sufficient to allow the concluion that the lack of SU(VAR)3-7 affects significantly the levels of transcripts of seven X-linked genes. If they exist, changes were indeed expected to be very small. For MSLs mutations, the magnitude of the decrease is very modest considering the severe failure of dosage compensation (around 1.5). Taking into account that the Su(var)3-7 mutation induces only 30% lethality among males, expected changes in transcript accumulation are predicted to be even smaller. Moreover, transcripts analysis was done in male larvae and some slight biological variations between the three samples cannot be avoided though great care was taken on samples homogeneity. Finally, normalizing to internal autosomal genes RNA could also introduce a bias. It is believed that in the case, quantitative RT-PCR experiment was not the appropriate method to detect very small changes of expression (Spierer, 2008).

In consequence, an alternative system was used to test the implication of SU(VAR)3-7 on dosage compensation. The effect of increased or decreased Su(var)3-7 expression on the dosage compensated expression of the white gene carried by P transgenes was determined. Strikingly, it was observed that lack and excess of SU(VAR)3-7 decreases the white expression specifically in males, and never in females. This is a strong indication that the wild type dose of SU(VAR)3-7 is required for correct dosage compensated expression of the white gene. Interestingly, Su(var)3-7 over-expression affects white expression when the gene is localized on the X chromosome and not on autosomes, although white is still partially dosage compensated on autosomes. This may result from the combination of two phenomena: On the X chromosome, excess of SU(VAR)3-7 induces preferential enrichment of heterochromatic silencing proteins and partial loss of MSLs. On autosomes, heterochromatic proteins recruitment is less visible and, in addition, the MSLs are massively present. Consequently the dosage compensation of a P(white) transgene linked to the X chromosome is more likely to be perturbed by excess of SU(VAR)3-7 than an autosomal insertion (Spierer, 2008).

In sum, in this study has revealed a role for SU(VAR)3-7 on global X chromosome morphology with an impact on the distribution of MSLs proteins, thus highlighting the contribution of SU(VAR)3-7 to the intriguing issue of X specific DCC targeting. It appears also that SU(VAR)3-7 is required for the viability of dosage compensated flies and the expression of a dosage compensated X-linked gene, suggesting a puzzling interplay between heterochromatin and the DCC. SU(VAR)3-7 plays a subtle role on dosage compensation: Flies need SU(VAR)3-7, especially the maternal protein, for correct dosage compensation but, at the same time, excess of SU(VAR)3-7 has a negative effect on dosage compensation. Future interest will focus on the fascinating issue of the molecular nature of heterochromatin/DCC intersection (Spierer, 2008).

JIL-1 and Su(var)3-7 interact genetically and counteract each other's effect on position-effect variegation in Drosophila

The essential JIL-1 histone H3S10 kinase is a key regulator of chromatin structure that functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of the JIL-1 kinase, two of the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms. This study addresses the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. The lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene, and Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, in the absence of JIL-1 kinase activity, Su(var)3-7 gets redistributed and upregulated on the chromosome arms. Reducing the dose of the Su(var)3-7 gene dramatically decreases this redistribution; however, the spreading of H3K9me2 to the chromosome arms is unaffected, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality. These observations suggest a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity (Deng, 2010).

While Su(var)3-9, Su(var)3-7, and HP1a reciprocal interactions are well documented at pericentric regions they are not universal. For example, HP1 binding on the fourth chromosome has been shown to be independent of Su(var)3-9; a tethering system to recruit HP1a to euchromatic sites has shown that HP1a-mediated silencing can operate in a Su(var)3-9-independent manner. Moreover, evidence has been provided that at least two different molecular mechanisms regulate Su(var)3-9 localization, one dependent on HP1 and one dependent on the JIL-1 kinase. These findings indicate that although Su(var)3-9, Su(var)3-7, and HP1a cooperate in heterochromatin formation and gene silencing at pericentric chromosome sites, they may function independently at other regions such as the chromosome arms. This study shows that the lethality but not the chromosome morphology defects associated with the null JIL-1 phenotype to a large degree can be rescued by reducing the dose of the Su(var)3-7 gene. This effect was observed with three different alleles of Su(var)3-7, strongly suggesting it is likely to be specific to Su(var)3-7 and not to second site modifiers. Furthermore, evidence is provided that JIL-1 levels and/or activity regulate the chromosome localization of Su(var)3-7 and that Su(var)3-7 levels are dramatically redistributed to the chromosome arms in conjunction with a reduced presence at the chromocenter in the absence of JIL-1 (Deng, 2010).

Previously, it has been demonstrated that JIL-1 genetically interacts with Su(var)3-9 but not with Su(var)2-5, suggesting that the lethality and disruption of chromosome morphology observed when JIL-1 levels are decreased are associated with ectopic Su(var)3-9 activity on the chromosomal arms and unrelated to HP1a recruitment. In this scenario, the spreading of the H3K9me2 mark to ectopic locations on the chromosomes is likely to lead to heterochromatization and repression of gene expression at these sites, leading to increased lethality. This hypothesis has been supported by genetic interaction assays that demonstrate that the lethality of JIL-1 null mutants can be almost completely rescued by a reduction in Su(var)3-9 dosage that prevents ectopic dimethylation of histone H3K9. However, this study has shown that while reducing the dose of Su(var)3-7 also rescues viability of JIL-1 null mutant larvae, H3K9me2 in polytene squashes still spreads to the chromosome arms, strongly indicating that ectopic Su(var)3-9 activity is not a direct cause of lethality, but rather that Su(var)3-9-mediated recruitment of Su(var)3-7 is a necessary factor. Futhermore, since viability was rescued despite no obvious improvement in chromosome morphology, the lethality caused by loss of JIL-1 function is not likely to be a consequence of perturbed chromosome morphology. Taken together these observations give rise to a model where Su(var)3-7 functions as an effector downstream of Su(var)3-9 and H3K9 dimethylation in heterochromatic spreading and gene silencing that is normally counteracted by JIL-1 kinase activity. How Su(var)3-7 may mediate these effects is unknown and will require additional studies (Deng, 2010).

The inherent components of heterochromatin Su(var)3-9, HP1a, and Su(var)3-7 display a haplosuppressor/triploenhancer dosage-dependent effect on PEV. Additional copies of all three genes cause strong enhancement of white variegation in w^m4, and in genetic interaction tests, the suppressor effect of Su(var)3-9 null mutations dominates the triplo-dependent enhancer effect of Su(var)2-5) and Su(var)3-7. Furthermore, it has been recently demonstrated that the gain-of-function JIL-1^Su(var)3-1 allele is one of the strongest suppressors of PEV so far described at all the PEV arrangements that have been tested. This allele even counteracts gene repression that is caused by overexpression of the major determinants of heterochromatin formation, e.g., Su(var)3-9, Su(var)2-5, and Su(var)3-7. The JIL-1^Su(var)3-1 allele generates truncated proteins with COOH-terminal deletions that mislocalize to ectopic chromatin sites, leading to expanded histone H3S10 phosphorylation. On the basis of these findings, a model has been proposedfor a dynamic balance between euchromatin and heterochromatin, where as can be monitored in PEV arrangements, the boundary between these two states is determined by antagonistic functions of euchromatic regulators (JIL-1) and the determinants of heterochromatin assembly. This study has further tested this hypothesis using JIL-1 loss-of-function alleles which are shown can act as haploenhancers of PEV. This included PEV of the w^m4 allele where, interestingly, combinations of strong hypomorphic JIL-1 alleles act as suppressors, not enhancers. It has been proposed that this is due to a reduction in the levels of heterochromatic factors near the inversion breakpoint that reduces its potential for heterochromatic spreading and silencing. As predicted by this hypothesis it was shown that in chromosome squash preparations from JIL^z2/+ larvae there was no discernible redistribution of the H3K9me2 heterochromatic mark. It was further demonstrated that JIL-1 and Su(var)3-7 alleles can counteract each other's effect on PEV. In all three PEV arrangements tested, Su(var)3-7 loss-of-function alleles acted as strong haplosuppressors as indicated by a high proportion of nearly completely red eyes, whereas JIL-1 loss-of-function alleles acted as strong haploenhancers as indicated by a high proportion of flies with nearly completely white ommatidia. However, in double mutant backgrounds, variegation of the proportion of red ommatidia was substantively restored and closer to the distribution when wild-type levels of JIL-1 and Su(var)3-7 proteins were present. These results strongly support a genetic interaction between JIL-1 and Su(var)3-7 and provide evidence that a finely tuned balance between the levels of JIL-1 and Su(var)3-7 contributes to the regulation of PEV (Deng, 2010).

While several potential mechanisms for heterochromatin spreading and gene silencing have been identified, the concept of a dynamic balance between euchromatin and heterochromatin implies that euchromatic factors may have similar spreading potential. However, the mechanisms that actively may lead to the expansion of euchromatic domains have received comparatively less attention. In Drosophila, the studies of the JIL-1 kinase have demonstrated that histone H3S10 phosphorylation is an important epigenetic modification potentially regulating both the establishment and maintenance of euchromatin. For example, it has been shown using a LacI tethering system, that JIL-1-mediated ectopic H3S10 phosphorylation can cause a change in higher-order chromatin structure from a condensed heterochromatin-like state to a more open euchromatic state. Thus, spreading of JIL-1 activity has the potential to expand euchromatic domains and counteract gene silencing in heterochromatic regions. However, while it has been shown that the COOH-terminal region of JIL-1 can directly interact with the histone H3 tail region, it remains to be established how JIL-1 targeting to specific chromatin regions is regulated and how dynamic this regulation is (Deng, 2010).

Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation

Loss of Su(var)3-7 or HP1 suppresses the genomic silencing of position-effect variegation, whereas over-expression enhances it. In addition, loss of Su(var)3-7 results in preferential male lethality. In polytene chromosomes deprived of Su(var)3-7, a specific bloating of the male X chromosome is observed, leading to shortening of the chromosome and to blurring of its banding pattern. In addition, the chromocenter, where heterochromatin from all polytene chromosomes fuses, appears decondensed. The same chromosomal phenotypes are observed as a result of loss of HP1. Mutations of Su(var)3-7 or of Su(var)2-5, the gene encoding HP1, also cause developmental defects, including a spectacular increase in size of the prothoracic gland and its polytene chromosomes. Thus, although structurally very different, the two proteins cooperate closely in chromosome organization and development. Finally, bloating of the male X chromosome in the Su(var)3-7 mutant depends on the presence of a functional dosage compensation complex on this chromosome. This observation reveals a new and intriguing genetic interaction between epigenetic silencing and compensation of dose (Spierer, 2005).

Su(var)3-7 function is still poorly understood. It encodes a large protein associated with pericentric heterochromatin, telomeres and a few euchromatic sites on interphase polytene chromosomes. Seven widely spaced zinc fingers stand out in the sequence of the N-terminal half. In vitro, the zinc finger region of Su(var)3-7 has affinity for DNA, and preferentially for some satellite sequences. There is also evidence for direct binding of Su(var)3-7 with DNA in vivo. The N-terminal half of Su(var)3-7 interacts nonspecifically in vivo with heterochromatin and euchromatin, whereas the C-terminal half promotes interaction with itself, and with pericentric heterochromatin. Su(var)3-7 also interacts genetically and physically with HP1 and with Su(var)3-9, as determined in yeast by the two-hybrid assay and in vivo. To decipher the function of Su(var)3-7, mutants were generated by homologous recombination, and a detailed examination was undertaken of their phenotype. Su(var)3-7 was shown to be essential, the maternal contribution being sufficient for viability. Interestingly, males are more sensitive than females to the lack of Su(var)3-7. The cause of this lethality is unknown (Spierer, 2005).

This study reports the building of a new mutant of Su(var)3-7 by homologous recombination: described are the phenotypes of mutations on polytene chromosome morphology and on the organism -- these are similar to phenotypes resulting from mutational loss of HP1. The male X chromosome is more sensitive to these effects, leading to an understanding of an interaction between the modifier of PEV Su(var)3-7 and the dosage compensation machinery. It is concluded that the importance of the roles and partnership of Su(var)3-7 and HP1 extend beyond genomic silencing in the maintenance of chromosome integrity and function, including the male X-specific chromosome-wide mechanism of dosage compensation (Spierer, 2005).

Polytene chromosomes are affected similarly by severe loss of Su(var)3-7 or HP1. In both cases, the main mutant phenotype is a bloated X in males, and an expanded chromocenter in males and females. Why is chromosome morphology modified when HP1 or Su(var)3-7 amounts are strongly reduced? There are several possible explanations. Su(var)3-7 and HP1 are both required for stability of chromatid association, and reduction of dose could lead to dissociation. This mechanism has been suggested for similar phenotypes in other conditions. This hypothesis could be tested by determining whether a phenomenon based on chromatid association, such as transvection, is affected in Su(var)3-7 or HP1 mutants. Su(var)3-7 and HP1 are required for compaction of intercalary heterochromatin on euchromatic arms. The loss of this compaction, similar to what is seen at the chromocenter, could lead to bloating and disruption of the banding pattern. If indeed Su(var)3-7 and HP1 are instrumental in chromosome compaction, then one could expect that excess amounts of the proteins lead in turn to an excess of compaction. This is actually the case for Su(var)3-7; increasing amounts of Su(var)3-7 first affect the male X chromosome, which becomes strongly compacted. Furthermore, targeting HP1 to an ectopic site promotes chromosomal loops linking this ectopic site with sites of intercalary heterochromatin. The question remains of the particular sensitivity of the male X chromosome to loss and excess of Su(var)3-7 and to loss of HP1 (Spierer, 2005).

That the male X chromosome is affected first and most severely could result from association of this chromosome with the dosage compensation complex (DCC). Chromatin relaxation triggered by the DCC in the male X would render it more sensitive to variations of the amount of chromatin-associated proteins. Indeed, male X bloating and shortening has been observed in several conditions, and has been named the 'pompon' phenotype and described as resulting from specific environmental aggressions or mutations. Male X bloating was described as resulting from the loss of several chromatin-modifying factors such as Jil-1 or the Nurf complex. The various environmental and genetic conditions in which bloating of the male X occurs underline the peculiar sensitivity of the phenotype, and could explain the differences of phenotype intensity seen using different X chromosomes (Spierer, 2005).

Finally, the X-chromosome-specific phenotype might result from a direct interaction between the DCC and silencing factors. This paper indeed demonstrates a genetic interaction between an essential gene of the dosage compensation machinery, mle, and Su(var)3-7. However, in the wild type, preferential association of Su(var)3-7 with the polytene male X chromosome has not been detected using either a polyclonal antibody raised against Su(var)3-7 sequences, or a monoclonal antibody raised against the tag of HA-Su(var)3-7. However, preferential association with the male X is seen when Su(var)3-7 is over-expressed from a transgene. At this point it is not possible to distinguish between two possibilities: either Su(var)3-7 modulates the transcription level of the X chromosome by counteracting the DCC relaxing effect, or it protects the X-linked genes that do not need to be dosage compensated. The role of HP1 also remains to be explored. No preferential association of HP1 with the male X polytene chromosome has been seen. Nevertheless, when Su(var)3-7 is over-expressed, HP1 is found associated preferentially with the male X (Spierer, 2005).

In conclusion, Su(var)3-7 and HP1 participate in chromocenter and male X polytene chromosome integrity. The similarity of the phenotypes seen in mutations of either one, the partial compensation of the loss of dose in one by an increase of dose in the other in PEV, and the physical interaction between Su(var)3-7 and HP1 seen in vitro and in vivo (Delattre, 2000) all point to the same conclusion. These two structurally very different proteins cooperate closely in chromosome organization. An interaction also existes between Su(var)3-7 and compensation of dose. This interaction between the genomic silencing of PEV dependent on Su(var)3-7 association, and hyperactivation dependent on association of the DCC, needs to be unravelled (Spierer, 2005).

Increased expression of Drosophila Su(var)3-7 triggers Su(var)3-9-dependent heterochromatin formation

The Su(var)3-7 protein is essential for fly viability, and several lines of evidence support its key importance in heterochromatin formation: it binds to pericentric heterochromatin, it potently suppresses variegation and it interacts with HP1. However, the mode of action of Su(var)3-7 is poorly understood. This study investigate in vivo the consequences of increased Su(var)3-7 expression on fly viability and chromatin structure. A large excess of Su(var)3-7 induces lethality, whereas lower doses permit survival and cause spectacular changes in the morphology of polytene chromosomes in males, and to a lesser extent in females. The male X is always the most affected chromosome: it becomes highly condensed and shortened, and its characteristic banding pattern is modified. In addition, Su(var)3-7 was found over the complete length of all chromosomes. This event coincides with the appearance of heterochromatin markers such as histone H3K9 dimethylation and HP1 at many sites on autosomes and, more strikingly, on the male X chromosome. These two features are strictly dependent on the histone-methyltransferase Su(var)3-9, whereas the generalised localisation of Su(var)3-7 is not. These data provide evidence for a dose-dependent regulatory role of Su(var)3-7 in chromosome morphology and heterochromatin formation. Moreover they show that Su(var)3-7 expression is sufficient to induce Su(var)3-9-dependent ectopic heterochromatinisation and suggest a functional link between Su(var)3-7 and the histone-methyltransferase Su(var)3-9 (Delattre, 2004).

Central role of Drosophila SU(VAR)3-9, a Su(var)3-7 interactor, 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).

Functional dissection of the Drosophila modifier of variegation Su(var)3-7

An increase in the dose of the heterochromatin-associated Su(var)3-7 protein of Drosophila augments the genomic silencing of position-effect variegation. This study expressed a number of fragments of the protein in flies to assign functions to the different domains. Specific binding to pericentric heterochromatin depends on the C-terminal half of the protein. The N terminus, containing six of the seven widely spaced zinc fingers, is required for binding to bands on euchromatic arms, with no preference for pericentric heterochromatin. In contrast to the enhancing properties of the full-length protein, the N terminus half has no effect on heterochromatin-dependent position-effect variegation. In contrast, the C terminus moiety suppresses variegation. This dominant negative effect on variegation could result from association of the fragment with the wild type endogenous protein. Indeed, a domain of self-association was found, and it was mapped to the C-terminal half. Furthermore, a small fragment of the C-terminal region actually depletes pericentric heterochromatin from endogenous Su(var)3-7 and has a very strong suppressor effect. This depletion is not followed by a depletion of HP1, a companion of Su(var)3-7. This indicates that Su(var)3-7 does not recruit HP1 to heterochromatin. It is proposed in conclusion that the association of Su(var)3-7 to heterochromatin depends on protein-protein interaction mediated by the C-terminal half of the sequence, while the silencing function requires also the N-terminal half containing the zinc fingers (Jaquet, 2002; Full text of article).

The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1

Position-effect variegation results from mosaic silencing by chromosomal rearrangements juxtaposing euchromatin genes next to pericentric heterochromatin. An increase in the amounts of the heterochromatin-associated Su(var)3-7 and HP1 proteins augments silencing. Using the yeast two-hybrid protein interaction trap system, HP1 has been isolated using Su(var)3-7 as a bait. Three binding sites on Su(var)3-7 for HP1 have been delimited. On HP1, the C-terminal moiety, including the chromo shadow domain, is required for interaction. In vivo, both proteins co-localize not only in heterochromatin, but also in a limited set of sites in euchromatin and at telomeres. When delocalized to the sites bound by the protein Polycomb in euchromatin, HP1 recruits Su(var)3-7. In contrast with euchromatin genes, a decrease in the amounts of both proteins enhances variegation of the light gene, one of the few genetic loci mapped within pericentric heterochromatin. This body of data supports a direct link between Su(var)3-7 and HP1 in the genomic silencing of position-effect variegation (Delattre, 2000).

SU(VAR)3-7, a Drosophila heterochromatin-associated protein and companion of HP1 in the genomic silencing of position-effect variegation

An increase in the dose of the Su(var)3-7 locus of Drosophila enhances the genomic silencing of position-effect variegation caused by centromeric heterochromatin. This study shows that the product of Su(var)3-7 is a nuclear protein which associates with pericentromeric heterochromatin at interphase, whether on diploid chromosomes from embryonic nuclei or on polytene chromosomes from larval salivary glands. The protein also associates with the partially heterochromatic chromosome 4. As these phenotypes and localizations resemble those described by others for the Su(var)205 locus and its heterochromatin-associated protein HP1, the presumed co-operation of the two proteins was tested further. The effect of the dose of Su(var)3-7 on silencing of a number of variegating rearrangements and insertions is strikingly similar to the effect of the dose of Su(var)205 reported by others. In addition, the two loci interact genetically, and the two proteins co-immunoprecipitate from nuclear extracts. The results suggest that SU(VAR)3-7 and HP1 co-operate in building the genomic silencing associated with heterochromatin (Cléard, 1997).

Search PubMed for articles about Drosophila Su(var)3-7

Beckstead, R. B., et al. (2005). Bonus, a Drosophila TIF1 homolog, is a chromatin-associated protein that acts as a modifier of position-effect variegation. Genetics 169: 783-794. PubMed ID: 15545640

Cléard, F., Delattre, M. and Spierer, P. (1997). SU(VAR)3-7, a Drosophila heterochromatin-associated protein and companion of HP1 in the genomic silencing of position-effect variegation. Embo J. 16: 5280-5288. PubMed ID: 9311988

Delattre, M., Spierer, A., Tonka, C. H. and Spierer, P. (2000). The genomic silencing of position-effect variegation in Drosophila melanogaster: interaction between the heterochromatin-associated proteins Su(var)3-7 and HP1. J. Cell. Sci. 113 Pt 23: 4253-4261. PubMed ID: 11069770

Delattre, M., Spierer, A., Jaquet, Y. and Spierer, P. (2004). Increased expression of Drosophila Su(var)3-7 triggers Su(var)3-9-dependent heterochromatin formation. J. Cell. Sci. 117: 6239-6247. PubMed ID: 15564384

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(4): 1183-92. PubMed ID: 20457875

Jaquet, Y., Delattre, M., Spierer, A. and Spierer, P. (2002). Functional dissection of the Drosophila modifier of variegation Su(var)3-7. Development 129(17): 3975-82. PubMed ID: 12163401

Johansson, A. M., Stenberg, P., Bernhardsson, C. and Larsson, J. (2007). Painting of fourth and chromosome-wide regulation of the 4th chromosome in Drosophila melanogaster. Embo J 26: 2307-2316. PubMed ID: 17318176

Reuter, G., Giarre, M., Farah, J., Gausz, J. and Spierer, A., et al. (1990). Dependence of position-effect variegation in Drosophila on dose of a gene encoding an unusual zinc-finger protein. Nature 344: 219-223. PubMed ID: 2107402

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. PubMed ID: 11867540

Seum, C., et al. (2002). Isolation of Su(var)3-7 mutations by homologous recombination in Drosophila melanogaster. Genetics 161: 1125-1136. PubMed ID: 12136016

Spierer, A., Seum, C., Delattre, M. and Spierer, P. (2005). Loss of the modifiers of variegation Su(var)3-7 or HP1 impacts male X polytene chromosome morphology and dosage compensation. J. Cell Sci. 118: 5047-5057. PubMed ID: 16234327

Spierer, A., Begeot, F., Spierer, P. and Delattre, M. (2008). SU(VAR)3-7 links heterochromatin and dosage compensation in Drosophila. PLoS Genet. 4(5): e1000066. PubMed ID: 18451980

Weiler, K. S. and Wakimoto, B. T. (1995). Heterochromatin and gene expression in Drosophila. Annu. Rev. Genet. 29: 577-605. PubMed ID: 8825487

date revised: 20 June 2009

Home page: The Interactive Fly © 2008 Thomas Brody, Ph.D.

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