A Trans-Acting Regulatory Gene That Inversely Affects the Expression of the White, Brown and Scarlet Loci in Drosophila

A trans-acting regulatory gene, Inr-a, that alters the level of expression of the white eye color locus as an inverse function of the number of its functional copies is described. Several independent lines of evidence demonstrate that this regulatory gene interacts with white via the promoter sequences. Among these are the observations that the inverse regulatory effect is conferred to the Adh gene when fused to the white promoter and that cis-regulatory mutants of white fail to respond. The phenotypic response to Inr-a is found in all tissues in which white is expressed, and mutants of the regulator exhibit a recessive lethality during larval periods. Increased white messenger RNA levels in pupal stages are found in Inr-a/+ individuals versus +/+ and a coordinate response is observed for mRNA levels from the brown and scarlet loci. All are structurally related and participate in pigment deposition. These experiments demonstrate that a single regulatory gene can exert an inverse effect on a target structural locus, a situation postulated from segmental aneuploid studies of gene expression and dosage compensation.     

Heterochromatic Trans-Inactivation of Drosophila White Transgenes

Position effect variegation of most Drosophila melanogaster genes, including the white eye pigment gene, is recessive. We find that this is not always the case for white transgenes. Three examples are described in which a lesion causing variegation is capable of silencing the white transgene on the paired homologue (trans-inactivation). These examples include two different transgene constructs inserted at three distinct genomic locations. The lesions that cause variegation of white minimally disrupt the linear order of genes on the chromosomes, permitting close homologous pairing. At one of these sites, trans-inactivation has also been extended to include a vital gene in the vicinity of the white transgene insertion. These findings suggest that many Drosophila genes, in many positions in the genome, can sense the heterochromatic state of a paired homologue.     

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.     

Comparative analysis of position-effect variegation mutations in Drosophila melanogaster delineates the targets of modifiers.

In Drosophila melanogaster, heterochromatin-induced silencing or position-effect variegation (PEV) of a reporter gene has provided insights into the properties of heterochromatin. Class I modifiers suppress PEV, and class II modifiers enhance PEV when the modifier gene is present in fewer than two doses. We have examined the effects of both class I and class II modifiers on four PEV mutations. These mutations include the inversions In(1)w(m4) and In(2R)bw(VDe2), which are classical chromosomal rearrangements that typify PEV mutations. The other mutations are a derivative of brown(Dominant), in which brown+ reporters are inactivated by a large block of heterochromatin, and a P[white+] transposon insertion associated with second chromosome heterochromatin. In general, we find that class I modifiers affect both classical and nonclassical PEV mutations, whereas class II modifiers affect only classical PEV mutations. We suggest that class II modifiers affect chromatin architecture in the vicinity of reporter genes, and only class I modifiers identify proteins that are potentially involved in heterochromatin formation or maintenance. In addition, our observations support a model in which there are different constraints on the process of heterochromatin-induced silencing in classical vs. nonclassical PEV mutations.     

Towards an understanding of position effect variegation

Most variegating position effects are a consequence of placing a euchromatic gene adjacent to alpha-heterochromatin. In such rearrangements, the affected locus is inactivated in some cells, but not others, thereby giving rise to a mosaic tissue of mutant and wild-type cells. A detailed examination of the molecular structure of three variegating white mottled mutations of Drosophila melanogaster, all of which are inversions of the X chromosome, reveals that their euchromatic breakpoints are clustered and located approximately 25 kb downstream of the white promoter and that the heterochromatic sequences to which the white locus is adjoined are transposons. An analysis of three revertants of the wm4 mutation, created by relocating white to another euchromatic site, demonstrates that they also carry some heterochromatically derived sequences with them upon restoration of the wild-type phenotype. This suggests that variegation is not controlled from a heterochromatic sequence immediately adjacent to the variegating gene but rather from some site more internal to the heterochromatic domain itself. As a consequence of this observation we have proposed a boundary model for understanding how heterochromatic domains may be formed. It has been recognized for many years that the phenotype of variegating position effects may be altered by the presence of trans-acting dominant mutations that act to either enhance or suppress variegation. Using P-element mutagenesis, we have induced and examined 12 dominant enhancers of variegation that represent four loci on the second and third chromosomes. Most of these mutations are cytologically visible duplications or deficiencies. They exert their dominant effects through changes in the copy number of wild-type genes and can be divided into two reciprocally acting classes. Class I modifiers are genes that act as enhancers of variegation when duplicated and as suppressors when mutated or deficient. Conversely, class II modifiers are genes that enhance when mutated or deleted and suppress when duplicated. The available data indicate that, in Drosophila, there are 20-30 loci capable of dominantly modifying variegation. Of these, most appear to be of the class I type whereas only two class II modifiers have been identified so far.(ABSTRACT TRUNCATED AT 400 WORDS)     

Krüppel homolog (Kr h) is a dosage-dependent modifier of gene expression in Drosophila

A lethal mutation in the Krüppel homolog (Kr h) was isolated in screens of P-element insertion mutations for modifiers of white gene expression. The mutation occurs in the 5' untranslated region of the Kr h gene and causes a lightening of the eye colour for several alleles of white due to a decrease in white steady-state mRNA levels at pupal stages. Two related genes, scarlet and brown, were significantly affected as well in early pupae. Genetic analysis of different white alleles suggests that enhancer sequences are necessary for interaction with KR H. Thus, the Kr h gene is a member of the dosage-dependent hierarchy effective upon white.     

white+ transgene insertions presenting a dorsal/ventral pattern define a single cluster of homeobox genes that is silenced by the polycomb-group proteins in Drosophila melanogaster.

We used the white gene as an enhancer trap and reporter of chromatin structure. We collected white+ transgene insertions presenting a peculiar pigmentation pattern in the eye: white expression is restricted to the dorsal half of the eye, with a clear-cut dorsal/ventral (D/V) border. This D/V pattern is stable and heritable, indicating that phenotypic expression of the white reporter reflects positional information in the developing eye. Localization of these transgenes led us to identify a unique genomic region encompassing 140 kb in 69D1-3 subject to this D/V effect. This region contains at least three closely related homeobox-containing genes that are constituents of the iroquois complex (IRO-C). IRO-C genes are coordinately regulated and implicated in similar developmental processes. Expression of these genes in the eye is regulated by the products of the Polycomb-group (Pc-G) and trithorax-group (trx-G) genes but is not modified by classical modifiers of position-effect variegation. Our results, together with the report of a Pc-G binding site in 69D, suggest that we have identified a novel cluster of target genes for the Pc-G and trx-G products. We thus propose that ventral silencing of the whole IRO-C in the eye occurs at the level of chromatin structure in a manner similar to that of the homeotic gene complexes, perhaps by local compaction of the region into a heterochromatin-like structure involving the Pc-G products.     

Inactivation of reporter genes by cloned heterochromatic repeats of Drosophila melanogaster is accompanied by chromatin compaction

Cloned Stellate heterochromatic repeats caused unstable mosaic inactivation (position effect variegation; PEV) of the reporter gene mini-white. A number of known protein modifiers of the classical position effect induced by large heterochromatin blocks do not affect the expression of mini-white. This raises the question as to the specificity of chromatin compaction around the reporter gene. The inactivation of the mini-white gene has been found to be accompanied by a decrease in its methylation catalyzed by Escherichia coli dam-methyltransferase expressed in the genome of Drosophila. However, no changes in the nucleosome organization of mini-white have been found.     

Dosage-dependent modification of position-effect variegation in Drosophla

Many loci in Drosophila exhibit dosage effects on single phenotypes. In the case of modifiers of position-effect variegation, increases and decreases in dosage can have opposite effects on variegating phenotypes. This is seemingly paradoxical: if each locus encodes a limiting gene product sensitive to dosage decreases, then increasing the dosage of any one should have no effect, because the others should remain limiting. An earlier model put forward to resolve this paradox suggested that dosage-dependent modifiers encode protein subunits of a macromolecular complex that is sensitive to mass action equilibrium conditions. Because chemical equilibria are dynamic, however, such hypothetical complexes will be unstable to an extent that is inconsistent with the known properties of molecules that make up chromatin. An alternative model accounts for the dosage effects in terms of interactions between structural proteins that bind at multiple linked sites. These might include indirect interactions occurring between regulatory proteins and genes for structural proteins or their protein products. The large number of direct and inverse regulatory genes which are known to exist in Drosophila could account for the apparent genetic complexity that is seen for modifiers of position-effect variegation and for other systems of phenotypic modification.     

Dosage-dependent modifiers of position effect variegation in Drosophila and a mass action model that explains their effect

Twelve dominant enhancers of position effect variegation, representing four loci on the second and third chromosomes of Drosophila melanogaster, have been induced by P-element mutagenesis. Instead of simple transposon insertions, seven of these mutations are cytologically visible duplications and three are deficiencies. The duplications define two distinct regions, each coinciding with a locus that also behaves as a dominant haplo-dependent suppressor of variegation. Conversely, two of the deficiencies overlap with a region that contains a haplo-dependent enhancer of variegation while duplications of this same region act to suppress variegation. The third deficiency defines another haplo-dependent enhancer. These data indicate that loci capable of modifying variegation do so in an antipodal fashion through changes in the wild-type gene copy number and may be divided into two reciprocally acting classes. Class I modifiers enhance variegation when duplicated or suppress variegation when deficient. Class II modifiers enhance when deficient but suppress when duplicated. From our data, and those of others, we propose that in Drosophila there are about 20 to 30 dominant loci that modify variegation. Most appear to be of the class I type whereas only two class II modifiers have been identified so far. From these observations we put forth a model, based on the law of mass action, for understanding how such suppressor-enhancer loci function. We propose that each class I modifier codes for a structural protein component of heterochromatin and their effects on variegation are a consequence of their dosage dependent influence on the extent of the assembly of heterochromatin at the chromosomal site of the position effect. It is further proposed that class II modifiers may inhibit the class I products directly, bind to hypothetical termination sites that define heterochromatin boundaries or promote euchromatin formation. Consistent with our mass action model we find that combining two enhancers together produce additive and not epistatic effects. Also, since different enhancers have different relative strengths on different variegating mutants, we suggest that heterochromatic domains are constructed by a combinatorial association of proteins. The mass action model proposed here is of general significance for any assembly driven reaction and has implications for understanding a wide variety of biological phenomena.(ABSTRACT TRUNCATED AT 400 WORDS)     

Partial Revertants of the Transposable Element-Associated Suppressible Allele White-Apricot in Drosophila Melanogaster: Structures and Responsiveness to Genetic Modifiers

The eye color phenotype of white-apricot (wa), a mutant allele of the white locus caused by the insertion of the transposable element copia into a small intron, is suppressed by the extragenic suppressor suppressor-of-white-apricot (su(wa] and enhanced by the extragenic enhancers suppressor-of-forked su(f] and Enhancer-of-white-apricot (E(wa]. Derivatives of wa have been analyzed molecularly and genetically in order to correlate the structure of these derivatives with their response to modifiers. Derivatives in which the copia element is replaced precisely by a solo long terminal repeat (sLTR) were generated in vitro and returned to the germline by P-element mediated transformation; flies carrying this allele within a P transposon show a nearly wild-type phenotype and no response to either su(f) or su(wa). In addition, eleven partial phenotypic revertants of wa were analyzed. Of these, one appears to be a duplication of a large region which includes wa, three are new alleles of su(wa), two are sLTR derivatives whose properties confirm results obtained using transformation, and five are secondary insertions into the copia element within wa. One of these, waR84h, differs from wa by the insertion of the most 3' 83 nucleotides of the I factor. The five insertion derivatives show a variety of phenotypes and modes of interaction with su[f) and su(wa). The eye pigmentation of waR84h is affected by su(f) and E(wa), but not su(wa). These results demonstrate that copia (as opposed to the interruption of white sequences) is essential for the wa phenotype and its response to genetic modifiers, and that there are multiple mechanisms for the alteration of the wa phenotype by modifiers.     

Heterochromatic DNA repeats in Drosophila and unusual gene silencing in yeast cells

The 1.25-kb heterochromatic Stellate repeats of Drosophila melanogaster are capable of stably persisting in transgenic constructs and silencing the white reporter gene (mosaic position effect variegation). This system reveals an unusual form of silencing, which is insensitive to known modifiers of position effect variegation. The unusual form of silencing was studied with yeast Saccharomyces cerevisiae, a simple eukaryotic model. To be transferred into yeast cells, the D. melanogaster Stellate repeats were cloned in the pYAC4 centromeric vector (CEN4, URA3, TRP1, HIS3). The HIS3 and/or URA3 genes could be inactive in plasmids consisting of pYAC4 and the Stellate insert in yeast cells. Deletion of D. melanogaster DNA from the plasmid was found to activate the URA3 and HIS3 genes. It was assumed that the genes were repressed rather than damaged in the presence of the Stellate repeats and that a new form of gene silencing was revealed in S. cerevisiae.     

Inactivation of Reporter Genes by Cloned Heterochromatic Repeats of Drosophila melanogaster is Accompanied by Chromatin Compaction

Cloned Stellate heterochromatic repeats caused unstable mosaic inactivation (position effect variegation; PEV) of the reporter genemini-white. A number of known protein modifiers of the classical position effect induced by large heterochromatin blocks do not affect the expression of mini-white. This raises the question as to the specificity of chromatin compaction around the reporter gene. The inactivation of themini-white gene has been found to be accompanied by a decrease in its methylation catalyzed by Escherichia coli dam-methyltransferase expressed in the genome of Drosophila. However, no changes in the nucleosome organization of mini-whitehave been found.