The Effect of Modifiers of Position-Effect Variegation on the Variegation of Heterochromatic Genes of Drosophila Melanogaster

Dominant modifiers of position-effect variegation of Drosophila melanogaster were tested for their effects on the variegation of genes normally located in heterochromatin. These modifiers were previously isolated as strong suppressors of the variegation of euchromatic genes and have been postulated to encode structural components of heterochromatin or other products that influence chromosome condensation. While eight of the modifiers had weak or no detectable effects, six acted as enhancers of light (lt) variegation. The two modifiers with the strongest effects on lt were shown to also enhance the variegation of neighboring heterochromatic genes. These results suggest that the wild-type gene products of some modifiers of position-effect variegation are required for proper expression of genes normally located within or near the heterochromatin of chromosome 2. We conclude that these heterochromatic genes have fundamentally different regulatory requirements compared to those typical of euchromatic genes.     

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)     

Competition between Different Variegating Rearrangements for Limited Heterochromatic Factors in Drosophila Melanogaster

Position effect variegation (PEV) results from the juxtaposition of a euchromatic gene to heterochromatin. In its new position the gene is inactivated in some cells and not in others. This mosaic expression is consistent with variability in the spread of heterochromatin from cell to cell. As many components of heterochromatin are likely to be produced in limited amounts, the spread of heterochromatin into a normally euchromatic region should be accompanied by a concomitant loss or redistribution of the protein components from other heterochromatic regions. We have shown that this is the case by simultaneously monitoring variegation of a euchromatic and a heterochromatic gene associated with a single chromosome rearrangement. Secondly, if several heterochromatic regions of the genome share limited components of heterochromatin, then some variegating rearrangements should compete for these components. We have examined this hypothesis by testing flies with combinations of two or more different variegating rearrangements. Of the nine combinations of pairs of variegating rearrangements we studied, seven showed nonreciprocal interactions. These results imply that many components of heterochromatin are both shared and present in limited amounts and that they can transfer between chromosomal sites. Consequently, even nonvariegation portions of the genome will be disrupted by re-allocation of heterochromatic proteins associated with PEV. These results have implications for models of PEV.     

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)     

The Role of Heterochromatin in the Expression of a Heterochromatic Gene, the Rolled Locus of Drosophila Melanogaster

Constitutive heterochromatic regions of chromosomes are those that remain condensed through most or all of the cell cycle. In Drosophila melanogaster, the constitutive heterochromatic regions, located around the centromere, contain a number of gene loci, but at a much lower density than euchromatin. In the autosomal heterochromatin, the gene loci appear to be unique sequence genes interspersed among blocks of highly repeated sequences. Euchromatic genes do not function well when brought into the vicinity of heterochromatin (position-effect variegation). We test the possibility that the blocks of centromeric heterochromatin provide an environment essential for heterochromatic gene function. To assay directly the functional requirement of autosomal heterochromatic genes to reside in heterochromatin, the rolled (rl) gene, which is normally located deep in chromosome 2R heterochromatin, was relocated within small blocks of heterochromatin to a variety of euchromatic positions by successive series of chromosomal rearrangements. The function of the rl gene is severely affected in rearrangements in which the rl gene is isolated in a small block of heterochromatin, and these position effects can be reverted by rearrangements which bring the rl gene closer to any large block of autosomal or X chromosome heterochromatin. There is some evidence that five other 2R heterochromatic genes are also affected among these rearrangements. These findings demonstrate that the heterochromatic genes, in contrast to euchromatic genes whose function is inhibited by relocation to heterochromatin, require proximity to heterochromatin to function properly, and they argue strongly that a major function of the highly repeated satellite DNA, which comprises most of the heterochromatin, is to provide this heterochromatic environment.     

cis-Acting determinants of heterochromatin formation on Drosophila melanogaster chromosome four

The heterochromatic domains of Drosophila melanogaster (pericentric heterochromatin, telomeres, and the fourth chromosome) are characterized by histone hypoacetylation, high levels of histone H3 methylated on lysine 9 (H3-mK9), and association with heterochromatin protein 1 (HP1). While the specific interaction of HP1 with both H3-mK9 and histone methyltransferases suggests a mechanism for the maintenance of heterochromatin, it leaves open the question of how heterochromatin formation is targeted to specific domains. Expression characteristics of reporter transgenes inserted at different sites in the fourth chromosome define a minimum of three euchromatic and three heterochromatic domains, interspersed. Here we searched for cis-acting DNA sequence determinants that specify heterochromatic domains. Genetic screens for a switch in phenotype demonstrate that local deletions or duplications of 5 to 80 kb of DNA flanking a transposon reporter can lead to the loss or acquisition of variegation, pointing to short-range cis-acting determinants for silencing. This silencing is dependent on HP1. A switch in transgene expression correlates with a switch in chromatin structure, judged by nuclease accessibility. Mapping data implicate the 1360 transposon as a target for heterochromatin formation. We propose that heterochromatin formation is initiated at dispersed repetitive elements along the fourth chromosome and spreads for approximately 10 kb or until encountering competition from a euchromatic determinant.     

Sex Chromosome Meiotic Drive in DROSOPHILA MELANOGASTER Males

In Drosophila melanogaster males, deficiency for X heterochromatin causes high X-Y nondisjunction and skewed sex chromosome segregation ratios (meiotic drive). Y and XY classes are recovered poorly because of sperm dysfunction. In this study it was found that X heterochromatic deficiencies disrupt recovery not only of the Y chromosome but also of the X and autosomes, that both heterochromatic and euchromatic regions of chromosomes are affected and that the "sensitivity" of a chromosome to meiotic drive is a function of its length. Two models to explain these results are considered. One is a competitive model that proposes that all chromosomes must compete for a scarce chromosome-binding material in Xh(-) males. The failure to observe competitive interactions among chromosome recovery probabilities rules out this model. The second is a pairing model which holds that normal spermiogenesis requires X-Y pairing at special heterochromatic pairing sites. Unsaturated pairing sites become gametic lethals. This model fails to account for autosomal sensitivity to meiotic drive. It is also contradicted by evidence that saturation of Y-pairing sites fails to suppress meiotic drive in Xh(- ) males and that extra X-pairing sites in an otherwise normal male do not induce drive. It is argued that meiotic drive results from separation of X euchromatin from X heterochromatin.     

Genetic Analysis of Stellate Elements of Drosophila Melanogaster

Repeated elements are remarkably important for male meiosis and spermiogenesis in Drosophila melanogaster. Pairing of the X and Y chromosomes is mediated by the ribosomal RNA genes of the Y chromosome and X chromosome heterochromatin, spermiogenesis depends on the fertility factors of the Y chromosome. Intriguingly, a peculiar genetic system of interaction between the Y-linked crystal locus and the X-linked Stellate elements seem to be also involved in male meiosis and spermiogenesis. Deletion of the crystal element of the Y, via an interaction with the Stellate elements of the X, causes meiotic abnormalities, gamete-genotype dependent failure of sperm development (meiotic drive), and deposition of protein crystals in spermatocytes. The current hypothesis is that the meiotic abnormalities observed in cry(-) males is due to an induced overexpression of the normally repressed Ste elements. An implication of this hypothesis is that the strength of the abnormalities would depend on the amount of the Ste copies. To test this point we have genetically and cytologically examined the relationship of Ste copy number and organization to meiotic behavior in cry(-) males. We found that heterochromatic as well as euchromatic Ste repeats are functional and that the abnormality in chromosome condensation and the frequency of nondisjunction are related to Ste copy number. Moreover, we found that meiosis is disrupted after synapsis and that cry-induced meiotic drive is probably not mediated by Ste.     

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.     

Histone Gene Multiplicity and Position Effect Variegation in DROSOPHILA MELANOGASTER

The histone genes of wild-type Drosophila melanogaster are reiterated 100–150 times per haploid genome and are located in the segment of chromosome 2 that corresponds to polytene bands 39D2-3 to E1-2. The influence of altered histone gene multiplicity on chromatin structure has been assayed by measuring modification of the gene inactivation associated with position effect variegation in genotypes bearing deletions of the 39D-E segment. The proportion of cells in which a variegating gene is active is increased in genotypes that are heterozygous for a deficiency that removes the histone gene complex. Deletions that remove segments adjacent to the histone gene complex have no effect on the expression of variegating genes. Suppression of position effect variegation associated with reduction of histone gene multiplicity applies to both X-linked and autosomal variegating genes. Position effects exerted by both autosomal and sex-chromosome heterochromatin were suppressible by deletions of the histone gene complex. The suppression was independent of the presence of the Y chromosome. A deficiency that deletes only the distal portion of the histone gene complex also has the ability to suppress position effect variegation. Duplication of the histone gene complex did not enhance position effect variegation. Deletion or duplication of the histone gene complex in the maternal genome had no effect on the extent of variegation in progeny whose histone gene multiplicity was normal. These results are discussed with respect to current knowledge of the organization of the histone gene complex and control of its expression.     

Genetic and molecular characterization of a variegating hsp70-acZ fusion gene in the euchromatic 31 B region of Drosophila melanogaster.

A hsp70-lacZ fusion gene introduced into Drosophila melanogaster at the euchromatic 31B region by Pelement transformation displayed a variegated expression with respect to the lacZ fusion protein in the salivary gland cells under heat-shock conditions. The variegation is also reflected by the chromosome puffing pattern. Subsequent transposition of the 31B P element to other euchromatic positions restored wild-type activity, that is, a nonvariegated phenotype. A lower developmental temperature reduced the amount of expression under heat-shock conditions, similar to genes undergoing position-effect variegation (PEV). However, other modifiers of PEV did not affect the expression pattern of the gene. These results show a novel euchromatic tissue-specific variegation that is not associated with classical heterochromatic PEV.     

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.     

Euchromatic inversions causing mosaic gene expression in Drosophila hydei: A study of forked and Zebra

In D. hydei two new mutants, In(1)f³ and IN(5)Z, show obvious mosaic gene expression. Their phenotypic expression is susceptible to the breeding temperature and to the addition of a supernumerary Y chromosome to the chromosome set. In this respect the mutants resemble standard cases of position-effect variegation based on the action of heterochromatin. However, since neither centromeric nor sex chromosomal heterochromatin apparently are involved, the mutations point to a new type of variegation provoked by euchromatic sections. The mosaic patterns of these mutants, in particular those of In(1)f³, will be described.