Position-effect variegation in Drosophila melanogaster X chromosome inversion with a breakpoint in a satellite block and its suppression in a secondary rearrangement

In(1LR)pn2a is a pericentric inversion with a euchromatic breakpoint in the 2E polytene region and a heterochromatic breakpoint in the right arm of the X chromosome. It is associated with position-effect variegation (PEV) of the pn, wapl, Pgd and other vital loci of the 2E region, which are relocated near the bulk of the X heterochromatin. Cytological analysis showed that the rearrangement brings the 1A–2E euchromatic segment directly into contact with a major portion of the h34 block, a heterochromatic region that is positively stained by the N-banding technique and contains the AAGAG satellite sequences. Molecular cloning revealed the presence of a new junction between euchromatin and AAGAG satellite sequences and demonstrated that the euchromatic breakpoint of In(1LR)pn2a lies in the vinculin gene. In the X ray-induced secondary rearrangement In(1LR)r30, consisting of a pericentric inversion superimposed on In(1LR)pn2a, the h34 material remains associated with the 2E region but is separated from the rest of the X heterochromatin. In this case, the pn, wapl and Pgd loci no longer variegate, suggesting that the satellite-containing h34 region is not able per se to induce detectable PEV on the adjacent euchromatic genes. Received in revised form: 15 June 1997 / Accepted: 16 September 1997     

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.     

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.     

Cis-Effects of Heterochromatin on Heterochromatic and Euchromatic Gene Activity in Drosophila Melanogaster

Chromosomal rearrangements that juxtapose heterochromatin and euchromatin can result in mosaic inactivation of heterochromatic and euchromatic genes. This phenomenon, position effect variegation (PEV), suggests that heterochromatic and euchromatic genes differ in their regulatory requirements. This report describes a novel method for mapping regions required for heterochromatic genes, and those that induce PEV of a euchromatic gene. P transposase mutagenesis was used to generate derivatives of a translocation that variegated for the light(+) (lt(+)) gene and carried the euchromatic white(+) (w(+)) gene on a transposon near the heterochromatin-euchromatin junction. Cytogenetic and genetic analyses of the derivatives showed that P mutagenesis resulted in deletions of several megabases of heterochromatin. Genetic and molecular studies showed that the derivatives shared a euchromatic breakpoint but differed in their heterochromatic breakpoint and their effects on seven heterochromatic genes and the w(+) gene. Heterochromatic genes differed in their response to deletions. The lt(+) gene was sensitive to the amount of heterochromatin at the breakpoint but the heterochromatic 40Fa gene was not. The severity of variegated w(+) phenotype did not depend on the amount of heterochromatin in cis, but varied with local heterochromatic environment. These data are relevant for considering mechanisms of PEV of both heterochromatic and euchromatic genes.     

Eu-heterochromatic rearrangements induce replication of heterochromatic sequences normally underreplicated in polytene chromosomes of Drosophila melanogaster

In polytene chromosomes of D. melanogaster the heterochromatic pericentric regions are underreplicated (underrepresented). In this report, we analyze the effects of eu-heterochromatic rearrangements involving a cluster of the X-linked heterochromatic (Xh) Stellate repeats on the representation of these sequences in salivary gland polytene chromosomes. The discontinuous heterochromatic Stellate cluster contains specific restriction fragments that were mapped along the distal region of Xh. We found that transposition of a fragment of the Stellate cluster into euchromatin resulted in its replication in polytene chromosomes. Interestingly, only the Stellate repeats that remain within the pericentric Xh and are close to a new eu-heterochromatic boundary were replicated, strongly suggesting the existence of a spreading effect exerted by the adjacent euchromatin. Internal rearrangements of the distal Xh did not affect Stellate polytenization. We also demonstrated trans effects exerted by heterochromatic blocks on the replication of the rearranged heterochromatin; replication of transposed Stellate sequences was suppressed by a deletion of Xh and restored by addition of Y heterochromatin. This phenomenon is discussed in light of a possible role of heterochromatic proteins in the process of heterochromatin underrepresentation in polytene chromosomes.     

Copy Number and Orientation Determine the Susceptibility of a Gene to Silencing by Nearby Heterochromatin in Drosophila

The classical phenomenon of position-effect variegation (PEV) is the mosaic expression that occurs when a chromosomal rearrangement moves a euchromatic gene near heterochromatin. A striking feature of this phenomenon is that genes far away from the junction with heterochromatin can be affected, as if the heterochromatic state ``spreads.'''' We have investigated classical PEV of a Drosophila brown transgene affected by a heterochromatic junction ~60 kb away. PEV was enhanced when the transgene was locally duplicated using P transposase. Successive rounds of P transposase mutagenesis and phenotypic selection produced a series of PEV alleles with differences in phenotype that depended on transgene copy number and orientation. As for other examples of classical PEV, nearby heterochromatin was required for gene silencing. Modifications of classical PEV by alterations at a single site are unexpected, and these observations contradict models for spreading that invoke propagation of heterochromatin along the chromosome. Rather, our results support a model in which local alterations affect the affinity of a gene region for nearby heterochromatin via homology-based pairing, suggesting an alternative explanation for this 65-year-old phenomenon.     

Studying Trans-Effects of Modifiers on the Position-Effect Variegation in a Set of Euchromatin–Heterochromatin Rearrangements in Drosophila melanogaster

The effects of suppressors of position-effect variegation were studied in a set of euchromatin–heterochromatin rearrangements of the X chromosome accompanied by inactivation of the gene wapl.The rearrangements differed from one another in the size of the heterochromatic block adjacent to euchromatin, with the euchromatin–heterochromatin border remaining unchanged. In one rearrangement (r20), the position effect caused by a small block of adjacent heterochromatin may be determined by its interaction with the neighboring main heterochromatic region of the X chromosome. Chromosome 3 (the RT chromosome) was found to have a strong suppressing effect on all rearrangements, irrespective of the amount of heterochromatin adjacent to euchromatin. Su-var(3)9, a known suppressor of the position-effect variegation, had a considerably weaker suppressing effect. The RT chromosome had the strongest suppressing effect on the rearrangement r20.     

A reexamination of spreading of position-effect variegation in the white-roughest region of Drosophila melanogaster

In Drosophila, heterochromatin causes mosaic silencing of euchromatic genes brought next to it by chromosomal rearrangements. Silencing has been observed to "spread": genes closer to the heterochromatic rearrangement breakpoint are silenced more frequently than genes farther away. We have examined silencing of the white and roughest genes in the variegating rearrangements In(1)w(m4), In(1)w(mMc), and In(1)w(m51b). Eleven stocks bearing these chromosomes differ widely in the strength of silencing of white and roughest. Stock-specific differences in the relative frequencies of inactivation of white and roughest were found that map to the white-roughest region or the adjacent heterochromatin. Most stock-specific differences did not correlate with gross differences in the heterochromatic content of the rearranged chromosomes; however, two stocks, In(1)w(m51b) and In(1)w(mMc), were found to have anomalous additional heterochromatin that may act in trans to suppress variegating alleles. In comparing different stocks, the frequency of silencing of the roughest gene, which is more distant from heterochromatin, does not correlate with the frequency of silencing of the more proximal white gene on the same chromosome, in contradiction to the expectation of models of continuous linear propagation of silencing. We frequently observed rough eye tissue that is pigmented, as though an active white gene is skipped.     

Trans-effect of modifiers on position-effect variegation in a set of euchromatin-heterochromatin rearrangements in Drosophila melanogaster]

The effects of suppressors of position-effect variegation were studied in a set of euchromatin-heterochromatin rearrangements of the X chromosome accompanied by inactivation of the gene wapl. The rearrangements differed from one another in the size of the heterochromatic block adjacent to euchromatin, with the euchromatin-heterochromatin border remaining unchanged. In one rearrangement (r20), the position effect caused by a small block of adjacent heterochromatin may be determined by its interaction with the neighboring main heterochromatic region of the X chromosome. Chromosome 3 (the RT chromosome) was found to have a strong suppressing effect on all rearrangements, irrespective of the amount of heterochromatin adjacent to euchromatin. Su-var(3)9, a known suppressor of the position-effect variegation, had a considerably weaker suppressing effect. The RT chromosome had the strongest suppressing effect on the rearrangement r20.     

Trans-inactivation: Repression in a wrong place

Trans-inactivation is the repression of genes on a normal chromosome under the influence of a rearranged homologous chromosome demonstrating the position effect variegation (PEV). This phenomenon was studied in detail on the example of brown^Dominant allele causing the repression of wild-type brown gene on the opposite chromosome. We have investigated another trans-inactivation-inducing chromosome rearrangement, In(2)A4 inversion. In both cases, brown^Dominant and In(2)A4, the repression seems to be the result of dragging of the euchromatic region of the normal chromosome into the heterochromatic environment. It was found that cis-inactivation (classical PEV) and trans-inactivation show different patterns of distribution along the chromosome and respond differently to PEV modifying genes. It appears that the causative mechanism of trans-inactivation is de novo heterochromatin assembly on euchromatic sequences dragged into the heterochromatic nuclear compartment. Trans-inactivation turns out to be the result of a combination of heterochromatin-induced position effect and the somatic interphase chromosome pairing that is widespread in Diptera.     

Position Effect Variegation in Drosophila Melanogaster: Relationship between Suppression Effect and the Amount of Y Chromosome

Position effect variegation results from chromosome rearrangements which translocate euchromatic genes close to the heterochromatin. The euchromatin-heterochromatin association is responsible for the inactivation of these genes in some cell clones. In Drosophila melanogaster the Y chromosome, which is entirely heterochromatic, is known to suppress variegation of euchromatic genes. In the present work we have investigated the genetic nature of the variegation suppressing property of the D. melanogaster Y chromosome. We have determined the extent to which different cytologically characterized Y chromosome deficiencies and Y fragments suppress three V-type position effects: the Y-suppressed lethality, the white mottled and the brown dominant variegated phenotypes. We find that: (1) chromosomes which are cytologically different and yet retain similar amounts of heterochromatin are equally effective suppressors, and (2) suppression effect is positively related to the size of the Y chromosome deficiencies and fragments that we tested. It increases with increasing amounts of Y heterochromatin up to 60-80% of the entire Y, after which the effect reaches a plateau. These findings suggest suppression is a function of the amount of Y heterochromatin present in the genome and is not attributable to any discrete Y region.     

Distorted heterochromatin replication in Drosophila melanogaster polytene chromosomes as a result of euchromatin-heterochromatin rearrangements

Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin--heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.     

Distorted heterochromatin replication in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> polytene chromosomes as a result of euchromatin-heterochromatin rearrangements

Studies of the position effect resulting from chromosome rearrangements in Drosophila melanogaster have shown that replication distortions in polytene chromosomes correlate with heritable gene silencing in mitotic cells. Earlier studies mostly focused on the effects of euchromatin-heterochromatin rearrangements on replication and silencing of euchromatic regions adjacent to the heterochromatin breakpoint. This review is based on published original data and considers the effect of rearrangements on heterochromatin: heterochromatin blocks that are normally underrepresented or underreplicated in polytene chromosomes are restored. Euchromatin proved to affect heterochromatin, preventing its underreplication. The effect is opposite to the known inactivation effect, which extends from heterochromatin to euchromatin. The trans-action of heterochromatin blocks on replication of heterochromatin placed within euchromatin is discussed. Distortions of heterochromatin replication in polytene chromosomes are considered to be an important characteristic associated with the functional role of the corresponding genome regions.     

pitkin(D), a novel gain-of-function enhancer of position-effect variegation, affects chromatin regulation during oogenesis and early embryogenesis in Drosophila

The vast majority of the >100 modifier genes of position-effect variegation (PEV) in Drosophila have been identified genetically as haplo-insufficient loci. Here, we describe pitkin(Dominant) (ptn(D)), a gain-of-function enhancer mutation of PEV. Its exceptionally strong enhancer effect is evident as elevated spreading of heterochromatin-induced gene silencing along euchromatic regions in variegating rearrangements. The ptn(D) mutation causes ectopic binding of the SU(VAR)3-9 heterochromatin protein at many euchromatic sites and, unlike other modifiers of PEV, it also affects stable position effects. Specifically, it induces silencing of white+ transgenes inserted at a wide variety of euchromatic sites. ptn(D) is associated with dominant female sterility. +/+ embryos produced by ptn(D)/+ females mated with wild-type males die at the end of embryogenesis, whereas the ptn(D)/+ sibling embryos arrest development at cleavage cycle 1-3, due to a combined effect of maternally provided mutant product and an early zygotic lethal effect of ptn(D). This is the earliest zygotic effect of a mutation so far reported in Drosophila. Germ-line mosaics show that ptn+ function is required for normal development in the female germ line. These results, together with effects on PEV and white+ transgenes, are consistent with the hypothesis that the ptn gene plays an important role in chromatin regulation during development of the female germ line and in early embryogenesis.     

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.