Reduced levels of Su(var)3-9 but not Su(var)2-5 (HP1) counteract the effects on chromatin structure and viability in loss-of-function mutants of the JIL-1 histone H3S10 kinase

It has recently been demonstrated that activity of the essential JIL-1 histone H3S10 kinase is a major regulator of chromatin structure and that it functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing. In the absence of JIL-1 kinase activity, the major heterochromatin markers histone H3K9me2 and HP1 spread in tandem to ectopic locations on the chromosome arms. In this study, we show that the lethality as well as some of 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)3-9 gene. This effect was observed with three different alleles of Su(var)3-9, strongly suggesting it is specific to Su(var)3-9 and not to second site modifiers. This is in contrast to similar experiments performed with alleles of the Su(var)2-5 gene that codes for HP1 in Drosophila where no genetic interactions were detectable between JIL-1 and Su(var)2-5. Taken together, these findings indicate that while Su(var)3-9 histone methyltransferase activity is 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 HP1.     

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 melanogaster enhances the genomic silencing of position-effect variegation caused by centromeric heterochromatin. Here we show 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)2-5 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)2-5 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.     

SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing

This review summarizes genetic, molecular and biochemical studies of the SU(VAR)3-9 protein and the evidence for its key role in heterochromatin formation and heterochromatic gene silencing. The Su(var)3-9 locus was first identified as a dominant modifier of position-effect variegation (PEV) in Drosophila melanogaster. Together with Su(var)2-5 and Su(var)3-7, Su(var)3-9 belongs to the group of haplo-suppressor loci which show a triplo-dependent enhancer effect. All three genes encode heterochromatin-associated proteins. Su(var)3-9 is epistatic to the PEV modifier effects of Su(var)2-5 and Su(var)3-7, and it also dominates the effect of the Y chromosome on PEV. These genetic data support a central role of the SU(VAR)3-9 protein in heterochromatic gene silencing, one that is correlated with its activity as a histone H3-K9 methyltransferase (HMTase). In fact, SU(VAR)3-9 is the main chromocenter-specific HMTase of Drosophila. SU(VAR)3-9 and HP1, the product of Su(var)2-5, are main constituents of heterochromatin protein complexes and the interaction between these two proteins is interdependent. Functional analysis in fission yeast, Drosophila and mammals demonstrate that SU(VAR)3-9-dependent gene silencing processes are conserved in these organisms. This is also demonstrated by the rescue of Drosophila Su(var)3-9 mutant phenotypes with human SUV39H1 transgenes.     

Heterochromatin protein 1 is involved in control of telomere elongation in Drosophila melanogaster

Telomeres of Drosophila melanogaster contain arrays of the retrotransposon-like elements HeT-A and TART. Their transposition to broken chromosome ends has been implicated in chromosome healing and telomere elongation. We have developed a genetic system which enables the determination of the frequency of telomere elongation events and their mechanism. The frequency differs among lines with different genotypes, suggesting that several genes are in control. Here we show that the Su(var)2-5 gene encoding heterochromatin protein 1 (HP1) is involved in regulation of telomere length. Different Su(var)2-5 mutations in the heterozygous state increase the frequency of HeT-A and TART attachment to the broken chromosome end by more than a hundred times. The attachment occurs through either HeT-A/TART transposition or recombination with other telomeres. Terminal DNA elongation by gene conversion is greatly enhanced by Su(var)2-5 mutations only if the template for DNA synthesis is on the same chromosome but not on the homologous chromosome. The Drosophila lines bearing the Su(var)2-5 mutations maintain extremely long telomeres consisting of HeT-A and TART for many generations. Thus, HP1 plays an important role in the control of telomere elongation in D. melanogaster.     

The large isoform of Drosophila melanogaster heterochromatin protein 2 plays a critical role in gene silencing and chromosome structure

Drosophila melanogaster heterochromatin protein 2 (HP2) interacts with heterochromatin protein 1 (HP1). In polytene chromosomes, HP2 and HP1 colocalize at the chromocenter, telomeres, and the small fourth chromosome. We show here that HP2 is present in the arms as well as the centromeric regions of mitotic chromosomes. We also demonstrate that Su(var)2-HP2 exhibits a dosage-dependent modification of variegation of a yellow reporter transgene, indicating a structural role in heterochromatin formation. We have isolated and characterized 14 new mutations in the Su(var)2-HP2 gene. Using wm4h, many (but not all) mutant alleles show dominant Su(var) activity. Su(var)2-HP2 mutant larvae show a wide variety of mitotic abnormalities, but not the telomere fusion seen in larvae deficient for HP1. The Su(var)2-HP2 gene codes for two isoforms: HP2-L (approximately 365 kDa) and HP2-S (approximately 175 kDa), lacking exons 5 and 6. In general, mutations that affect only the larger isoform result in more pronounced defects than do mutations common to both isoforms. This suggests that an imbalance between large and small isoforms is particularly deleterious. These results indicate a role for HP2 in the structural organization of chromosomes and in heterochromatin-induced gene silencing and show that the larger isoform plays a critical role in these processes.     

Recombinogenic effects of suppressors of position-effect variegation in Drosophila

Compact chromatin structure, induction of gene silencing in position-effect variegation (PEV), and crossing-over suppression are typical features of heterochromatin. To identify genes affecting crossing-over suppression by heterochromatin we tested PEV suppressor mutations for their effects on crossing over in pericentromeric regions of Drosophila autosomes. From the 46 mutations (28 loci) studied, 16 Su(var) mutations of the nine genes Su(var)2-1, Su(var)2-2, Su(var)2-5, Su(var)2-10, Su(var)2-14, Su(var)2-15, Su(var)3-3, Su(var)3-7, and Su(var)3-9 significantly increase in heterozygotes or by additive effects in double and triple heterozygotes crossing over in the ri-p(p) region of chromosome 3. Su(var)2-2(01) and Su(var)2-14(01) display the strongest recombinogenic effects and were also shown to enhance recombination within the light-rolled heterochromatic region of chromosome 2. The dominant recombinogenic effects of Su(var) mutations are most pronounced in proximal euchromatin and are accompanied with significant reduction of meiotic nondisjunction. Our data suggest that crossing-over suppression by heterochromatin is controlled at chromatin structure as well as illustrate the possible effects of heterochromatin on total crossing-over frequencies in the genome.     

Ectopic HP1 promotes chromosome loops and variegated silencing in Drosophila

A transgene inserted in euchromatin exhibits mosaic expression when targeted by a fusion protein made of the DNA-binding domain of GAL4 and the heterochromatin-associated protein HP1. The silencing responds to the loss of a dose of the dominant modifiers of position-effect variegation Su(var)3-7 and Su(var)2-5, the locus encoding HP1. The genomic environs of the insertion site at 87C1 comprise the dispersed repetitive elements micropia and alphagamma. In the presence of the GAL4-HP1 chimera, the polytene chromosomes of this line form loops between the insertion site of the transgene and six other sections of chromosome 3R, as well as, rarely, with pericentric and telomeric heterochromatin. In contrast to the insertion site of the transgene at 87C, the six loop-forming sites in the euchromatic arm were each previously described as intercalary heterochromatin. Moreover, GAL4-HP1 tethering on one homologue trans-inactivates the reporter on the other. HP1, probably together with other partners, could thus facilitate the coalescence of dispersed middle repetitive sequences, and organize the heterochromatic structure responsible for the variegated silencing of nearby euchromatic genes.     

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. Here we address the role of the third major heterochromatin component, the zinc-finger protein Su(var)3-7. We show 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 and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalancing effect on position-effect variegation (PEV). Furthermore, we show that 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 was 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.SU(VAR)3-9, a histone methyltransferase, Su(var)2-5, HP1a, and Su(var)3-7, a 1250-residue zinc-finger protein are all inherent components of pericentric heterochromatin (Rea et al. 2000; Eissenberg and Elgin 2000; Schotta et al. 2002; Delattre et al. 2004; Ebert et al. 2004) and are important factors for silencing of reporter genes by heterochromatic spreading in Drosophila (for review see Weiler and Wakimoto 1995; Girton and Johansen 2008). Su(var)3-9 has been shown to catalyze most of the dimethylation of the histone H3K9 residue which in turn can promote HP1a and Su(var)3-7 recruitment (Schotta et al. 2002; Jaquet et al. 2006). In addition, Su(var)3-9, HP1a, and Su(var)3-7 can directly interact with each other, suggesting a model where interdependent interactions between Su(var)3-9, HP1a, and Su(var)3-7 lead to heterochromatin assembly at pericentric sites (Lachner et al. 2001; Schotta et al. 2002; Elgin and Grewal 2003; Jaquet et al. 2006). Heterochromatin formation in Drosophila is initiated early in development through active removal of H3K4 methylation by the LSD1 demethylase homolog Su(var)3-3 (Rudolph et al. 2007). Subsequently, a developmentally regulated balance between Su(var)3-3 H3K4 demethylase, Su(var)3-9 H3K9 methyltransferase, and RPD3 H3K9 deacetylase activity contribute to conserve the distinction between euchromatic and heterochromatic domains (Rudolph et al. 2007). Thus, highly complex interactions between multiple heterochromatic and euchromatic factors are likely to contribute to the regulation of a dynamic balance between the distinct chromatin environments promoting gene activity and gene silencing.It has recently been demonstrated that activity of the essential JIL-1 histone H3S10 kinase (Jin et al. 1999; Wang et al. 2001) is a major regulator of chromatin structure (Deng et al. 2005; 2008) and that it functions to maintain euchromatic domains while counteracting heterochromatization and gene silencing (Ebert et al. 2004; Zhang et al. 2006; Lerach et al. 2006; Bao et al. 2007). In the absence of the JIL-1 kinase, the major heterochromatin markers H3K9me2 and HP1a spread in tandem to ectopic locations on the chromosome arms with the most pronounced increase on the X chromosomes (Zhang et al. 2006; Deng et al. 2007). However, overall levels of the H3K9me2 mark and HP1a were unchanged, suggesting that the spreading was accompanied by a redistribution that reduces the levels in pericentromeric heterochromatin. Genetic interaction assays demonstrated that the lethality as well as some of 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)3-9 gene (Zhang et al. 2006; Deng et al. 2007). This is in contrast to similar experiments performed with alleles of the Su(var)2-5 gene where no genetic interactions were detectable between JIL-1 and Su(var)2-5 (Deng et al. 2007) Thus, these findings indicate that while Su(var)3-9 histone methyltransferase activity may be a 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. However, the potential role of the third major heterochromatin component, Su(var)3-7, was not addressed in these studies. Here we show that Su(var)3-7, like Su(var)3-9, genetically interacts with JIL-1, that reducing the dose of Su(var)3-7 significantly reduces the lethality of JIL-1 null mutants, and that Su(var)3-7 and JIL-1 loss-of-function mutations have an antagonistic and counterbalacing effect on position-effect variegation (PEV).     

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo

HP1 is a small nonhistone chromosomal protein of Drosophila melanogaster predominantly localized to the pericentric heterochromatin. We have shown previously that mutations in the HP1 coding sequences are associated with dominant suppression of heterochromatic position-effect variegation, and with recessive lethality. When fused to an Hsp70 heat shock gene promoter, the cDNA encoding HP1 supports the heat shock-inducible accumulation of HPI protein in transgenic flies; this cDNA construct complements the dominant suppression of position-effect variegation associated with mutations in the HP1 gene. Here, we report experiments demonstrating that the heat shock-driven HP1 cDNA is capable of fully rescuing the recessive lethality associated with HP1 mutations in a heat shock-dependent fashion. If heat shock-induced HP1 expression is delayed for as long as 5 days, more than half of the mutant flies still survive until adulthood, consistent with a substantial maternal contribution to embryonic and larval viability. Elevating HP1 levels as late as 7–8 days of development is sufficient to enhance variegation three-fold, suggesting that the extent of heterochromatic position effect can be modified subsequent to the initial appearance of HP1 in the nuclei of syncytial blastoderm embryos.     

pR plasmid replication provides evidence that single-stranded DNA induces the SOS system in vivo

HP1 is a small nonhistone chromosomal protein of Drosophila melanogaster predominantly localized to the pericentric heterochromatin. We have shown previously that mutations in the HP1 coding sequences are associated with dominant suppression of heterochromatic position-effect variegation, and with recessive lethality. When fused to an Hsp70 heat shock gene promoter, the cDNA encoding HP1 supports the heat shock-inducible accumulation of HPI protein in transgenic flies; this cDNA construct complements the dominant suppression of position-effect variegation associated with mutations in the HP1 gene. Here, we report experiments demonstrating that the heat shock-driven HP1 cDNA is capable of fully rescuing the recessive lethality associated with HP1 mutations in a heat shock-dependent fashion. If heat shock-induced HP1 expression is delayed for as long as 5 days, more than half of the mutant flies still survive until adulthood, consistent with a substantial maternal contribution to embryonic and larval viability. Elevating HP1 levels as late as 7–8 days of development is sufficient to enhance variegation three-fold, suggesting that the extent of heterochromatic position effect can be modified subsequent to the initial appearance of HP1 in the nuclei of syncytial blastoderm embryos.     

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

In this study, we show that 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, which is a major catalyst for dimethylation of the histone H3K9 residue, HP1 recruitment, and the formation of silenced heterochromatin. We further provide evidence 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, we propose a model where JIL-1 kinase activity functions to maintain euchromatic regions by antagonizing Su(var)3-9-mediated heterochromatization.     

Silencing at Drosophila telomeres: nuclear organization and chromatin structure play critical roles.

Transgenes inserted into the telomeric regions of Drosophila melanogaster chromosomes exhibit position effect variegation (PEV), a mosaic silencing characteristic of euchromatic genes brought into juxtaposition with heterochromatin. Telomeric transgenes on the second and third chromosomes are flanked by telomeric associated sequences (TAS), while fourth chromosome telomeric transgenes are most often associated with repetitious transposable elements. Telomeric PEV on the second and third chromosomes is suppressed by mutations in Su(z)2, but not by mutations in Su(var)2-5 (encoding HP1), while the converse is true for telomeric PEV on the fourth chromosome. This genetic distinction allowed for a spatial and molecular analysis of telomeric PEV. Reciprocal translocations between the fourth chromosome telomeric region containing a transgene and a second chromosome telomeric region result in a change in nuclear location of the transgene. While the variegating phenotype of the white transgene is suppressed, sensitivity to a mutation in HP1 is retained. Corresponding changes in the chromatin structure and inducible activity of an associated hsp26 transgene are observed. The data indicate that both nuclear organization and local chromatin structure play a role in this telomeric PEV.     

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 homologues, 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 chromocentre 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, SU(VAR)3-9-HP1 interaction is interdependent and governs distinct localization patterns of both proteins. In Su(var)3-9 null mutants, concentration of HP1 at the chromocentre 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 dispersed 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.     

kurtz, a novel nonvisual arrestin, is an essential neural gene in Drosophila

The kurtz gene encodes a novel nonvisual arrestin. krz is located at the most-distal end of the chromosome 3R, the third gene in from the telomere. krz is expressed throughout development. During early embryogenesis, krz is expressed ubiquitously and later is localized to the central nervous system, maxillary cirri, and antennal sensory organs. In late third instar larvae, krz message is detected in the fat bodies, the ventral portion of the thoracic-abdominal ganglia, the deuterocerebrum, the eye-antennal imaginal disc, and the wing imaginal disc. The krz(1) mutation contains a P-element insertion within the only intron of this gene and results in a severe reduction of function. Mutations in krz have a broad lethal phase extending from late embryogenesis to the third larval instar. The fat bodies of krz(1) larva precociously dissociate during the midthird instar. krz(1) is a type 1 melanotic tumor gene; the fat body is the primary site of melanotic tumor formation during the third instar. We have functionally rescued these phenotypes with both genomic and cDNA transgenes. Importantly, the expression of a full-length krz cDNA within the CNS rescues the krz(1) lethality. These experiments establish the krz nonvisual arrestin as an essential neural gene in Drosophila.