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.     

Position effect variegation of the mosaic type, arising as a result of transposition AR4-24P[white, rosy] in the Drosophila melanogaster genome

A line with the mosaic expression of the white+ transgene was obtained by inducing transposition of the AR4-24P[white, rosy] transposon and was used for the second round of induction. As a result, 57 lines with the mosaic eye pigmentation were obtained. In situ hybridization and Southern blotting showed that genomic DNA fragments flanking AR4-24 were, in some cases, transposed together with the transposon. A spontaneous loss of these fragments resulted in reversion to the wild-type phenotype. The mosaic eye pigmentation in a line that carried the AR4-24 transposon flanked with the same fragments in region 24D1-2 was not affected by the Su(var)3-6 gene modifying position effect variegation (PEV). Other PEV modifiers, Su(var)3-9 and Su(var)2-5, had only a slight effect on PEV; Su(var)3-7 restored the wild-type phenotype. The genomic fragments captured by the transposon may contain DNA sequences that autonomously induce mosaic PEV of the white gene.     

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.     

E(var)3-9 of Drosophila melanogaster encodes a zinc finger protein

The importance of a gene's natural chromatin environment for its normal expression is poignantly illustrated when a change in chromosome position results in variable gene repression, such as is observed in position effect variegation (PEV) when the Drosophila melanogaster white (omega) gene is juxtaposed with heterochromatin. The Enhancer of variegation 3-9 [E(var)3-9] gene was one of over a hundred loci identified in screens for mutations that dominantly modify PEV. Haploinsufficiency for E(var)3-9 enhances omegam4 variegation, as would be expected from increased heterochromatin formation. To clarify the role of E(var)3-9 in chromosome structure, the gene has been cloned and its mutant alleles characterized. The involvement of E(var)3-9 in structure determination was supported by its reciprocal effects on euchromatic and heterochromatic PEV; E(var)3-9 mutations increased expression of a variegating heterochromatic gene in two tissue types. E(var)3-9 mutations also had a recessive phenotype, maternal effect lethality, which implicated E(var)3-9 function in an essential process during embryogenesis. Both phenotypes of E(var)3-9 mutations were consistent with its proposed function in promoting normal chromosome structure. The cloning of E(var)3-9 by classical genetic methods revealed that it encodes a protein with multiple zinc fingers, but otherwise novel sequence.     

Modifiers of terminal deficiency-associated position effect variegation in Drosophila

Terminal deletions of a Drosophila minichromosome (Dp(1;f)1187) dramatically increase the position effect variegation (PEV) of a yellow(+) body-color gene located in cis. Such terminal deficiency-associated PEV (TDA-PEV) can be suppressed by the presence of a second minichromosome, a phenomenon termed "trans-suppression." We performed a screen for mutations that modify TDA-PEV and trans-suppression. Seventy suppressors and enhancers of TDA-PEV were identified, but no modifiers of trans-suppression were recovered. Secondary analyses of the effects of these mutations on different PEV types identified 10 mutations that modify only TDA-PEV and 6 mutations that modify TDA-PEV and only one other type of PEV. One mutation, a new allele of Su(var)3-9, affects all forms of PEV, including silencing associated with the insertion of a transgene into telomeric regions (TPE). This Su(var)3-9 allele is the first modifier of PEV to affect TPE and provides a unique link between different types of gene silencing in Drosophila. The remaining mutations affected multiple PEV types, indicating that general PEV modifiers impact TDA-PEV. Modifiers of TDA-PEV may identify proteins that play important roles in general heterochromatin biology, including proteins involved in telomere structure and function and the organization of chromosomes in the interphase nucleus.     

Position-effect variegation and the genetic dissection of chromatin regulation in Drosophila

In position-effect variegation (PEV) genes become silenced by heterochromatisation. Genetic dissection of this process has been performed by means of dominant suppressor [Su(var)] and enhancer [E(var)] mutations. Selective genetic screens allowed mass isolation of more than 380 PEV modifier mutations identifying about 150 genes. Genetic fine structure studies revealed unique dosage dependent effects. Most of the haplo-dependent Su(var) and E(var) genes do not display triplo-dependent effects. Several Su(var) loci with triplo-dependent opposite enhancer effects have been identified and shown to encode heterochromatin-associated proteins. From these the evolutionary conserved histone H3 lysine 9 methyltransferase SU(VAR)3-9 plays a central role in heterochromatic gene silencing. Molecular function of most PEV modifier genes is still unknown also including genes identified with mutations displaying lethal interaction to heterochromatin. Their analysis should contribute to further understanding of processes connected with regulation of higher order chromatin structure and epigenetic programming.     

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.     

The transposon A(R)4-24P[white, rosy] in Drosophila melanogaster is subject to position-effect variegation at a non-centromeric insertion site

The white gene within the transposon A(R)4-24P[white,rosy] inserted at cytological location 24D1-2 in the euchromatic portion of the Drosophila melanogaster genome exhibits a mosaic pattern of expression which is modified by temperature and Y-chromosome number, as in cases of classical position-effect variegation (PEV). The eye colour of the flies in this variegated stock remains mosaic in the presence of the PEV modifier Su(var)3-6, slightly less so with Su(var)3-9 and Su(var)2-5, and full suppression of variegation occurs in the presence of Su(var)3-7. We have induced further transposition of A(R)4-24 and isolated two mosaic stocks with this transgene at new cytological locations. In these stocks, the A(R)4-24 transposon was flanked by the same genomic DNA fragments as in the original location. Spontaneous loss of these fragments leads to reversion of the variegated eye colour to wild-type. We suggest that the flanking DNA fragments from 24D1-2 are capable of inducing position-effect variegation without any association with centromeric heterochromatin. In situ hybridisation and Southern analysis demonstrate that the 5' flanking genomic fragment contains repeated sequences which are abundantly present in heterochromatin.     

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.     

A Cytogenetic and Genetic Characterization of a Group of Closely Linked Second Chromosome Mutations That Suppress Position-Effect Variegation in Drosophila Melanogaster

Characterization of a group of dominant second chromosome suppressor of position-effect variegation (PEV) (Su(var)) mutants has revealed a variety of interesting properties, including: maternal-effect suppression of PEV, homozygous lethality or semilethality and male-specific hemizygous lethality, female infecundity, acute sensitivity to the amount of heterochromatin in the cell and sensitivity to sodium butyrate. Deficiency/duplication mapping and complementation tests have revealed that eight of the mutants define at least two genes in section 31 of the left arm of chromosome 2 and they suggest that a ninth corresponds to an additional nonessential Su(var) gene within or near this region. The effects of specific deficiencies and a duplication on PEV indicate that the expression of one or more of the Su(var) genes in this region of the chromosome is dose-dependent, i.e., capable of haplo-abnormal suppression and triplo-abnormal enhancement. Interestingly, the appearance of certain visible phenotypes among a subset of the mutants suggests that they may possess antimorphic properties. Our results are consistent with the hypothesis that two of these Su(var) genes encode structural components of heterochromatin. We also report that two previously isolated mutants located in 31E and 31F-32A act as recessive suppressors of PEV.     

A high proportion of genes involved in position effect variegation also affect chromosome inheritance

Suppressors and enhancers of position effect variegation (PEV) have been linked to the establishment and maintenance of heterochromatin. The presence of centromeres and other inheritance elements in heterochromatic regions suggests that suppressors and enhancers of PEV, Su(var) s and E(var)s [collectively termed Mod(var)s], may be required for chromosome inheritance. In order to test this hypothesis, we screened 59 ethyl methanesulfonate-generated Drosophila Mod(var)s for dominant effects on the partially compromised inheritance of a minichromosome (J21A) missing a portion of the genetically defined centromere. Nearly half of these Mod(var)s significantly increased or decreased the transmission of J21A. Analyses of homozygous mutant larval neuroblasts suggest that these mutations affect cell cycle progression and native chromosome morphology. Five out of six complementation groups tested displayed mitotic abnormalities, including phenotypes such as telomere fusions, overcondensed chromosomes, and low mitotic index. We conclude that Mod(var)s as a group are highly enriched for genes that encode essential inheritance functions. We propose that a primary function of Mod(var)s is to promote chromosome inheritance, and that the gene silencing phenotype associated with PEV may be a secondary consequence of the heterochromatic structures required to carry out these functions.Communicated by R. AllshireH.D. Le and K.M. Donaldson contributed equally to this work     

Enhancer of Polycomb is a suppressor of position-effect variegation in Drosophila melanogaster.

Polycomb group (PcG) genes of Drosophila are negative regulators of homeotic gene expression required for maintenance of determination. Sequence similarity between Polycomb and Su(var)205 led to the suggestion that PcG genes and modifiers of position-effect variegation (PEV) might function analogously in the establishment of chromatin structure. If PcG proteins participate directly in the same process that leads to PEV, PcG mutations should suppress PEV. We show that mutations in E(Pc), an unusual member of the PcG, suppress PEV of four variegating rearrangements: In(l)wm4, B(SV), T(2;3)Sb(V) and In(2R)bw(VDe2). Using reversion of a Pelement insertion, deficiency mapping, and recombination mapping as criteria, homeotic effects and suppression of PEV associated with E(Pc) co-map. Asx is an enhancer of PEV, whereas nine other PcG loci do not affect PEV. These results support the conclusion that there are fewer similarities between PcG genes and modifiers of PEV than previously supposed. However, E(Pc) appears to be an important link between the two groups. We discuss why Asx might act as an enhancer of PEV.     

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.     

Genetic factors controlling white gene expression of the transposon A(R) 4-24 at a telomere in Drosophila melanogaster.

The position effect of the AR 4-24 P[white, rosy] transposon was studied at cytological position 60F. Three copies of the transposon (within approximately 50-kb region) resulted in a spatially restricted pattern of white variegation. This pattern was modified by temperature and by removal of the Y chromosome, suggesting that it was due to classical heterochromatin-induced position effect variegation (PEV). In contrast with classical PEV, extra dose of the heterochromatin protein 1 (HP1) suppressed white variegation and one dose enhanced it. The effect of Pc-G, trx-G, and other PEV suppressors was also tested. It was found that E(Pc)1, TrlR85, and mutations of Su(z)2C relieve A(R) 4-24-silencing and z1 enhances it. To explain the results obtained with these modifiers, it is proposed that PEV and telomeric position effect can counteract each other at this particular cytological site.