Position-effect variegation in Drosophila: the modifier Su(var)3-7 is a modular DNA-binding protein.

An increase in the dose of the Su(var)3-7 locus of Drosophila augments heterochromatin-promoted variegated silencing. The deduced protein sequence of Su(var)3-7 reveals seven widely spaced zinc fingers. We found that Su(var)3-7 has affinity for DNA in vitro and that the minimal protein sequence requirement for DNA binding is any module containing two zinc fingers and the interval between them. As Su(var)3-7 is a heterochromatin-associated protein, we tested its affinity for various satellite DNA sequences in vitro. The AATAT and 353-bp elements have the highest affinity. If affinity for satellite DNAs contributes to the presence of Su(var)3-7 in heterochromatin, a general affinity for DNA, or sequences yet to be determined, suggests a function in the genomic silencing of position-effect variegation: expansion of heterochromatin, whether continuous by spreading or discontinuous by pairing with sequence elements scattered through euchromatin, could use the affinity of Su(var)3-7 for DNA.

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.

The bithorax complex of Drosophila melanogaster: Underreplication and morphology in polytene chromosomes.

The level of polyteny of the Drosophila salivary gland chromosomes was determined throughout the chromosome region 89E1-4, the locus of the Bithorax Complex. A zone of underreplication spans the 300 kb of DNA from the Ubx to Abd-B loci. From the centromere proximal end of the complex, a 70-kb-long gradual decrease of polytenization starts with the Ubx transcription unit and, after a floor corresponding to the abd-A locus, raises gradually back to the maximum over 70 kb in the region of the Abd-B transcription unit. In flies carrying the mutation Suppressor of DNA Underreplication [Su(UR)ES], the underreplication of the Bithorax Complex is fully suppressed. In the wild type, the Bithorax Complex forms a weak point featuring thinner bands separated by clefts or constrictions. In Su(UR)ES strain in contrast, the 89E1-4 band looks like a single solid band consisting of homogenous dense material. We speculate that the wild-type Su(UR)ES protein hampers DNA replication of silenced domains and leads to their underreplication in salivary gland polytene chromosomes.

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, we have isolated HP1 using Su(var)3-7 as a bait. We have then delimited three binding sites on Su(var)3-7 for HP1. On HP1, the C-terminal moiety, including the chromo shadow domain, is required for interaction. In vivo, both proteins co-localise not only in heterochromatin, but also in a limited set of sites in euchromatin and at telomeres. When delocalised to the sites bound by the protein Polycomb in euchromatin, HP1 recruits Su(var)3-7. Finally, and 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.

A GAL4-HP1 fusion protein targeted near heterochromatin promotes gene silencing.

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.

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.

The histone deacetylase RPD3 counteracts genomic silencing in Drosophila and yeast.

Both position-effect variegation (PEV) in Drosophila and telomeric position-effect in yeast (TPE) result from the mosaic inactivation of genes relocated next to a block of centromeric heterochromatin or next to telomeres. In many aspects, these phenomena are analogous to other epigenetic silencing mechanisms, such as the control of homeotic gene clusters, X-chromosome inactivation and imprinting in mammals, and mating-type control in yeast. Dominant mutations that suppress or enhance PEV are thought to encode either chromatin proteins or factors that directly affect chromatin structure. We have identified an insertional mutation in Drosophila that enhances PEV and reduces transcription of the gene in the eye-antenna imaginal disc. The gene corresponds to that encoding the transcriptional regulator RPD3 in yeast, and to a human histone deacetylase. In yeast, RRD3-deletion strains show enhanced TPE, suggesting a conserved role of the histone deacetylase RPD3 in counteracting genomic silencing. This function of RPD3, which is in contrast to the general correlation between histone acetylation and increased transcription, might be due to a specialized chromatin structure at silenced loci.

The dose of a putative ubiquitin-specific protease affects position-effect variegation in Drosophila melanogaster.

A dominant insertional P-element mutation enhances position-effect variegation in Drosophila melanogaster. The mutation is homozygous, viable, and fertile and maps at 64E on the third chromosome. The corresponding gene was cloned by transposon tagging. Insertion of the transposon upstream of the open reading frame correlates with a strong reduction of transcript level. A transgene was constructed with the cDNA and found to have the effect opposite from that of the mutation, namely, to suppress variegation. Sequencing of the cDNA reveals a large open reading frame encoding a putative ubiquitin-specific protease (Ubp). Ubiquitin marks various proteins, frequently for proteasome-dependent degradation. Ubps can cleave the ubiquitin part from these proteins. We discuss the link established here between a deubiquitinating enzyme and epigenetic silencing processes.

Position-effect variegation in Drosophila depends on dose of the gene encoding the E2F transcriptional activator and cell cycle regulator.

A dominant mutation due to the insertion of a P-element at 93E on the third chromosome of Drosophila melanogaster enhances position-effect variegation. The corresponding gene was cloned by transposon tagging and the sequence of the transcript revealed that it corresponds to the gene encoding the transcriptional activator and cell cycle regulator dE2F. The transposon-tagged allele is homozygous viable, and the insertion of the transposon in an intron correlates with a strong reduction in the amount of transcript. A homozygous lethal null allele was found to behave as a strong enhancer when heterozygous. Overexpression of the gene in transgenic flies has the opposite effect of suppressing variegation. A link is established here, and discussed, between the dose of a transcriptional activator, which controls the cell cycle, and epigenetic silencing of chromosomal domains in Drosophila.

The modifier of position-effect variegation Suvar(3)7 of Drosophila: there are two alternative transcripts and seven scattered zinc fingers, each preceded by a tryptophan box.

An increase in the number of copies of the Drosophila locus Suvar(3)7 enhances position-effect variegation, i.e. the inactivation in some cells of genes brought close to heterochromatin by a chromosomal rearrangement. The locus produces two transcripts of 5047 and 4203 nt that differ solely by the length of their 3' untranslated region. That these transcripts encode the modifier of variegation Suvar(3)7 is demonstrated by genetic transformation with the corresponding cDNAs. The deduced protein is 1169 amino acids long and contains seven widely spaced zinc fingers. These fingers are each preceded at 11-16 amino acids before the N-terminal cysteine by a tryptophan-containing motif. The transcripts are maternally transmitted, but are also found throughout development. The ubiquitous distribution of transcripts in embryos and the different sequence motifs support our speculation that the locus encodes a chromosomal protein implicated in heterochromatin-mediated DNA silencing.

Position effect variegation and chromatin proteins.

Variegated phenotypes often result from chromosomal rearrangements that place euchromatic genes next to heterochromatin. In such rearrangements, the condensed structure of heterochromatin can spread into euchromatic regions, which then assume the morphology of heterochromatin and become transcriptionally inactive. In position-effect variegation (PEV) therefore, gene inactivation results from a change in chromatin structure. PEV has been intensively investigated in the fruitfly Drosophila, where the phenomenon allows a genetic dissection of chromatin components. Consequently, many genes have been identified which, when mutated, act as dominant modifiers (suppressors or enhancers) of PEV. Data available already demonstrate that genetic, molecular and developmental analysis of these genes provides an avenue to the identification of regulatory and structural chromatin components, and hence to fundamental aspects of chromosome structure and function.

Minigene rescues acetylcholinesterase lethal mutations in Drosophila melanogaster.

The gene encoding acetylcholinesterase in Drosophila melanogaster is over 34,000 base-pairs long. We have constructed a 5800 base-pair minigene containing 1500 base-pairs of genomic sequence upstream from the transcription start spliced to the coding sequence, but lacking the nine introns. After germline genetic transformation, this minigene rescues acetylcholinesterase lethal mutants. Tissue-specific distribution appears normal. This allows us to test site-directed mutations of acetylcholinesterase. In a first effort, deletion of most of the unusual 1000 bases leader and its intriguing short open reading frames showed no effect on gene expression. The way is open to study in vivo the structure-function relationships of acetylcholinesterase and insecticide resistance.

Dependence of position-effect variegation in Drosophila on dose of a gene encoding an unusual zinc-finger protein.

Position-effect variegation is the inactivation in some cells of a gene translocated next to heterochromatin, the region of the chromosome that is permanently condensed. The number of copies of the Drosophila gene Suvar(3)7 is a dose-limiting factor in this phenomenon, and seems from its sequence that it encodes a protein with five widely spaced zinc-fingers. This novel arrangement of zinc-fingers could help in packaging the chromatin fibre into heterochromatin, and also reflect a novel method of controlling the expression from DNA domains.

Drosophila melanogaster acetylcholinesterase gene. Structure, evolution and mutations.

Acetylcholinesterase is a key component of cholinergic neurotransmission. In Drosophila melanogaster, acetylcholinesterase is encoded by the Ace locus. We have determined the complete organization of the locus. The transcription unit is 34 kb (1 kb = 10(3) bases) long and encompasses ten exons. We have mapped the 5' end of the transcript, sequenced all the intron/exon boundaries, as well as the 3' end of the transcript. The deduced mature transcript is 4291 nucleotides long without poly(A). Sequencing of the promoter region reveals a potential TATA box and (GA)n motives. The Drosophila coding sequence is more split than its vertebrate counterparts, but the splicing sites of the two last exons are precisely conserved among Drosophila and vertebrate cholinesterases, and intriguingly also with the bovine thyroglobulin gene. Finally, a number of the mutations isolated in earlier genetic work are precisely placed on our molecular map in introns, exons and promoter regions. Among them, for example, a short deletion known to affect acetylcholinesterase level and tissue distribution removes promoter regions and the first non-coding exon.

Conservation of neural nicotinic acetylcholine receptors from Drosophila to vertebrate central nervous systems.

Nicotinic acetylcholine receptors (nAChR) are found both in vertebrate and insect central nervous systems. We have isolated a Drosophila gene by crosshybridization with a vertebrate probe. Structural conservation of domains of the deduced protein and of intron/exon boundaries indicate that the Drosophila gene encodes an nAChR alpha-like subunit (ALS). That the Drosophila gene product most resembles the neuronal set of vertebrate nAChRs alpha-subunits is also indicated by the failure of an ALS-beta-galactosidase fusion protein to bind alpha-bungarotoxin on blots in contrast to vertebrate endplate alpha-subunit constructions. The ALS encoding gene exceeds 54 kb in length and the transcript has a very long and unusual 5' leader. As we found previously for a gene whose product is also involved in cholinergic synapses, acetylcholinesterase, the leader encodes short open reading frames, which might be involved in translation control. We also note the presence of opa repeats in the gene, as has been found for various Drosophila genes expressed in the nervous system.

Modifiers of position-effect variegation in the region from 86C to 88B of the Drosophila melanogaster third chromosome.

Four dominant suppressor and one enhancer of variegation loci were mapped in the polytene chromosome region extending from section 86C to section 88B of the Drosophila melanogaster third chromosome using a set of deficiencies. The suppressor locus Su-var(3)14 maps in 86CD, Su-var(3)13 in 86F4-7, Su-var(3)6 in 87B4-7 and Su-var(3)7 in 87E4-5. The enhancer locus E-var(3)3 maps in 87E12-F11. Su-var(3)13, Su-var(3)6 and Su-var(3)7 are also defined by point mutant alleles originally identified by other criteria (Reuter et al. 1986). Duplications covering the suppressor loci Su-var(3)14, Su-var(3)13, Su-var(3)6 and Su-var(3)7 were found to reduce considerably the haplo-abnormal effect of heterozygous point mutants of the corresponding loci. One suppressor locus, Su-var(3)7, maps within a region which has previously been cloned. The positions of deficiency breakpoints delimiting the suppressor locus indicate that all the necessary sequences for its function are located within 10 kb of cloned DNA.

The Ace locus of Drosophila melanogaster: structural gene for acetylcholinesterase with an unusual 5' leader.

The Ace locus of Drosophila melanogaster has been mapped at the molecular level. cDNA clones from the locus have been isolated and their sequence determined, confirming that Ace forms the structural gene for acetylcholinesterase (AChE). The cDNAs have a 1950 nucleotide open reading frame from which the complete amino acid sequence of AChE has been deduced. The Drosophila enzyme is found to have extensive homology to the known sequence of Torpedo AChE. Ace cDNAs have an unusual structure with a long 5' leader and several short upstream open reading frames.

A gene required for the specification of dorsal-ventral pattern in Drosophila appears to encode a serine protease.

The maternal effect gene snake is required for the establishment of the dorsal-ventral axis during the embryonic development of Drosophila. The molecular cloning of the gene and analysis of a complementary DNA sequence suggest that the gene encodes a serine protease which is structurally similar to proteases involved in blood clotting, peptide processing, and complement fixation pathways.

Genes and loops in 320,000 base-pairs of the Drosophila melanogaster chromosome.

We have mapped the DNA sequences bound to the nuclear scaffold along 320,000 base-pairs of a genetically well-defined region of the Drosophila chromosome. We have found that the domains delimited by the scaffold attachment regions are heterogeneous in size (ranging from 26,000 to 112,000 base-pairs in this interval), and that the attachment sites are within unique sequences as judged by blot hybridization. We also found that looped domains contain up to five, or even eight, unrelated genes including, in some cases, more than one transcribed gene. The loop organization unravelled here in cultured cells does not correspond to the banding pattern seen in salivary gland polytene chromosomes.

Molecular Genetics of the ROSY-ACE Region of DROSOPHILA MELANOGASTER.

Three hundred and fifteen kilobases of DNA from the rosy-Ace region on chromosome 3R of D. melanogaster have previously been cloned and extensively characterized. We describe the isolation of nine new deficiency mutants that break within the 315-kb interval. The position of these breakpoints on the DNA map was determined by in situ and Southern hybridization. Further, we more precisely mapped the breakpoints of several deletions previously analyzed. The results permit us to delimit sequences essential to the known complementation groups in the region within approximately 20 kb in most cases. However, one gene, B16-1, is shown to contain essential sequences that span about 50 kb. Also, we demonstrate by overlapping deficiencies that a 45-kb DNA segment from the region, which includes one known complementation group, allows limited survival when deleted.