SU(VAR)3-7, a Drosophila heterochromatinassociated protein and companion of HP1 in the genomic silencing of position-effect variegation of homeotic gene complexes (i.e. Paro and Hogness, 1991; Fabienne Clé ard, Marion Delattre and Dorn et al., 1993; Fauvarque and Dura, 1993; Farkas Pierre Spierer 1 Tschiersch et al., 1994).Department of Zoology and Animal Biology, University of Geneva,The genetic modifiers of PEV could identify components 30 quai Ernest-Ansermet, CH-1211 Geneva 4, Switzerland of the mechanisms of genomic silencing (Eissenberg, 1989;Reuter and Spierer, 1992). In Drosophila melanogas-1 Corresponding author ter, haplo-suppressor loci with a triplo-enhancer effect on PEV are candidates for being structural components of An increase in the dose of the Su(var)3-7 locus of the heterochromatin conformation that spreads silencing Drosophila melanogaster enhances the genomic silencon euchromatic genes. Among these, we have cloned the ing of position-effect variegation caused by centromeric modifier locus Su(var)3-7, and shown that an increase in heterochromatin. Here we show that the product of its dose enhances PEV ; Cléard et al., Su(var)3-7 is a nuclear protein which associates 1995). The deduced protein sequence predicts that it with pericentromeric heterochromatin at interphase, encodes a large protein with seven widely separated zinc whether on diploid chromosomes from embryonic fingers. Another candidate, HP1, initially isolated as a nuclei or on polytene chromosomes from larval salivary heterochromatin-associated protein (James and Elgin, glands. The protein also associates with the partially 1986), later was found to be encoded by the haploheterochromatic chromosome 4. As these phenotypes suppressor triplo-enhancer of PEV Su(var)2-5 (Eissenberg and localizations resemble those described by others for et al., 1990, 1992). In polytene chromosomes, HP1 was the Su(var)2-5 locus and its heterochromatin-associated found to associate with centromeric heterochromatin and protein HP1, the presumed co-operation of the two chromosome 4, and to a lesser extent with a subset of proteins was tested further. The effect of the dose of telomeres and a few euchromatic bands (James et al., Su(var)3-7 on silencing of a number of variegating 1989). In embryonic nuclei, HP1 co-localizes with centrorearrangements and insertions is strikingly similar to meric heterochromatin in interphase chromosomes the effect of the dose of Su(var)2-5 reported by others. (Kellum et al., 1995). The involvement of HP1 in the In addition, the two loci interact genetically, and genomic silencing of PEV is also supported cytologically the two proteins co-immunoprecipitate from nuclear by its association with euchromatic loci inactivated by extracts. The results suggest that SU(VAR)3-7 and HP1PEV (Belyaeva et al., 1993). co-operate in building the genomic silencing associated It has been proposed that the heterochromatic conformwith heterochromatin.ation spreads silence by co-operative assembly of a variety Keywords: Drosophila/genom...

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, we observe a specific bloating of the male X chromosome, 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)37 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

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. Here we 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)37 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

Mammalian G9a is a euchromatic histone H3 lysine 9 (H3K9) methyltransferase essential for development. Here, we characterize the Drosophila homolog of G9a, dG9a. We generated a dG9a deletion allele by homologous recombination. Analysis of this allele revealed that, in contrast to recent findings, dG9a is not required for fly viability.

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.

We have constructed a new reporter transgene, Winkelried, equipped with a synthetic binding site for the yeast GAL4 transcriptional activator. The binding site is inserted between the white and lacZ reporter genes, and is flanked by FRT sequences. These elements allow excision of the GAL4 binding site by crossing the transgenic line with an FLP recombinase producing strain. We have generated by X-ray irradiation two independent chromosomal rearrangements, Heidi and Tell, relocating Winkelried next to pericentromeric heterochromatin. These rearrangements induce variegation of both white and lacZ. Variegation of Winkelried in the rearranged transgenic lines responds to the loss and excess of doses of the dominant suppressors of position-effect variegation (PEV) Su(var)3-7 and Su(var)2-5. Winkelried therefore constitutes a unique tool to test the effect on variegation in cis of any factor fused to the GAL4 DNA binding domain. Indeed, a chimeric protein, made of the DNA binding site of GAL4 and of HP1, the modifier of PEV encoded by Su(var)2-5, is shown to enhance variegation of Heidi and Tell. Excision of the binding sites for GAL4 in the variegating rearrangements Heidi and Tell abolishes the modifier effect of the GAL4-HP1 chimera. Therefore, in the Heidi and Tell rearrangements, enhancement of position-effect variegation depends strictly both on the concentration of GAL4-HP1 and on the presence of its binding site in the vicinity of the reporter genes.

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 modi®ers 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 ag. 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 loopforming 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.

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. We previously found that the morphology of this chromosome is sensitive to the amounts of the heterochromatin-associated protein SU(VAR)3-7. In this study, we examine the impact of change in levels of SU(VAR)3-7 on dosage compensation. We first demonstrate 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, we show 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.