Cell-by-cell dissection of gene expression and chromosomal interactions reveals consequences of nuclear reorganization

The functional consequences of long-range nuclear reorganization were studied in a cell-by-cell analysis of gene expression and long-range chromosomal interactions in the Drosophila eye and eye imaginal disk. Position-effect variegation was used to stochastically perturb gene expression and probe nuclear reorganization. Variegating genes on rearrangements of Chromosomes X, 2, and 3 were probed for long-range interactions with heterochromatin. Studies were conducted only in tissues known to express the variegating genes. Nuclear structure was revealed by fluorescence in situ hybridization with probes to the variegating gene and heterochromatin. Gene expression was determined alternately by immunofluorescence against specific proteins and by eye pigment autofluorescence. This allowed cell-by-cell comparisons of nuclear architecture between cells in which the variegating gene was either expressed or silenced. Very strong correlations between heterochromatic association and silencing were found. Expressing cells showed a broad distribution of distances between variegating genes and their own centromeric heterochromatin, while silenced cells showed a very tight distribution centered around very short distances, consistent with interaction between the silenced genes and heterochromatin. Spatial and temporal analysis of interactions with heterochromatin indicated that variegating genes primarily associate with heterochromatin in cells that have exited the cell cycle. Differentiation was not a requirement for association, and no differences in association were observed between cell types. Thus, long-range interactions between distal chromosome regions and their own heterochromatin have functional consequences for the organism.     

A structural basis for variegating position effects

Variegating position effects in Drosophila result from chromosome rearrangements where normal genes, having been placed next to heterochromatin, are inactivated in some cells but not in others, thereby producing a variegated tissue. We have determined that the euchromatic breakpoints for three variegating white mutants are clustered and lie approximately 25 kb downstream of the white structural gene. In each case the white locus is adjoined in the heterochromatin to a mobile genetic element. Satellite sequences are not involved. We also demonstrate that revertants of the variegating mutant, wm4, are reinversions that leave the initial wm4-heterochromatic junction intact so that some heterochromatin-derived sequences remain joined to white at its new location. These results suggest a simple model for understanding the structure of heterochromatic domains and how variegating position effects may arise.     

Functional properties of the heterochromatic sequences inducing w m4 position-effect variegation in Drosophila melanogaster

In position-effect variegation euchromatic genes are brought into the vicinity of heterochromatic sequences as a result of chromosomal rearrangements. This results in the inactivation of these genes in a proportion of cells causing a variegated phenotype. Tartof et al. (1984) have shown that the flanking heterochromatin in the w ^m4 variegating rearrangement in Drosophila melanogaster is homologous to the Type I inserts found in some portions of the rDNA repeats. We have studied the functional properties of these sequences using 51 revertant chromosomes, several variant lines of w ^m4 , strong enhancer mutations of position-effect variegation and X heterochromatin deletions. Our results suggest an array of tandemly repeated sequences showing additive effects and probably subject to magnification and reduction in number. Since only 3 of the 51 revertants isolated do not show variegation if strong enhancer mutations are introduced, only a very short sequence must be essential for the induction of white gene inactivation in w ^m4 . This suggests that the heterochromatic junction itself is sufficient to initiate the variegation of an adjacent gene. Parental source as well as paternal effects on the activity of these sequences have been detected. Revertant chromosomes of w ^m4 can be found after P-directed mutagenesis in hybrid dysgenic crosses suggesting mobile genetic elements at the breakpoints of inversion w ^m4 . These results are discussed with respect to the structural basis of positioneffect variegation as well as the function of certain heterochromatic sequences.     

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.     

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.     

P-Element repression in Drosophila melanogaster by variegating clusters of P-lacZ-white transgenes.

In Drosophila, clusters of P transgenes (P-lac-w) display a variegating phenotype for the w marker. In addition, X-ray-induced rearrangements of chromosomes bearing such clusters may lead to enhancement of the variegated phenotype. Since P-lacZ transgenes in subtelomeric heterochromatin have some P-element repression abilities, we tested whether P-lac-w clusters also have the capacity to repress P-element activity in the germline. One cluster (T-1), located on a rearranged chromosome (T2;3) and derived from a line bearing a variegating tandem array of seven P-lac-w elements, partially represses the dysgenic sterility (GD sterility) induced by P elements. This cluster also strongly represses in trans the expression of P-lacZ elements in the germline. This latter suppression shows a maternal effect. Finally, the combination of variegating P-lac-w clusters and a single P-lacZ reporter inserted in subtelomeric heterochromatic sequences at the X chromosome telomere (cytological site 1A) leads to strong repression of dysgenic sterility. These results show that repression of P-induced dysgenic sterility can be elicited in the absence of P elements encoding a polypeptide repressor and that a transgene cluster can repress the expression of a single homologous transgene at a nonallelic position. Implications for models of transposable element silencing are discussed.     

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.     

Genetics of P-element transposition into Drosophila melanogaster centric heterochromatin

Heterochromatin is a major component of higher eukaryotic genomes, but progress in understanding the molecular structure and composition of heterochromatin has lagged behind the production of relatively complete euchromatic genome sequences. The introduction of single-copy molecular-genetic entry points can greatly facilitate structure and sequence analysis of heterochromatic regions that are rich in repeated DNA. In this study, we report the isolation of 502 new P-element insertions into Drosophila melanogaster centric heterochromatin, generated in nine different genetic screens that relied on mosaic silencing (position-effect variegation, or PEV) of the yellow gene present in the transposon. The highest frequencies of recovery of variegating insertions were observed when centric insertions were used as the source for mobilization. We propose that the increased recovery of variegating insertions from heterochromatic starting sites may result from the physical proximity of different heterochromatic regions in germline nuclei or from the association of mobilizing elements with heterochromatin proteins. High frequencies of variegating insertions were also recovered when a potent suppressor of PEV (an extra Y chromosome) was present in both the mobilization and selection generations, presumably due to the effects of chromatin structure on P-element mobilization, insertion, and phenotypic selection. Finally, fewer variegating insertions were recovered after mobilization in females, in comparison to males, which may reflect differences in heterochromatin structure in the female and male germlines. FISH localization of a subset of the insertions confirmed that 98% of the variegating lines contain heterochromatic insertions and that these schemes produce a broader distribution of insertion sites. The results of these schemes have identified the most efficient methods for generating centric heterochromatin P insertions. In addition, the large collection of insertions produced by these screens provides molecular-genetic entry points for mapping, sequencing, and functional analysis of Drosophila heterochromatin.     

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.     

Cytogenetic analysis of the third chromosome heterochromatin of Drosophila melanogaster

Previous cytological analysis of heterochromatic rearrangements has yielded significant insight into the location and genetic organization of genes mapping to the heterochromatin of chromosomes X, Y, and 2 of Drosophila melanogaster. These studies have greatly facilitated our understanding of the genetic organization of heterochromatic genes. In contrast, the 12 essential genes known to exist within the mitotic heterochromatin of chromosome 3 have remained only imprecisely mapped. As a further step toward establishing a complete map of the heterochomatic genetic functions in Drosophila, we have characterized several rearrangements of chromosome 3 by using banding techniques at the level of mitotic chromosome. Most of the rearrangement breakpoints were located in the dull fluorescent regions h49, h51, and h58, suggesting that these regions correspond to heterochromatic hotspots for rearrangements. We were able to construct a detailed cytogenetic map of chromosome 3 heterochromatin that includes all of the known vital genes. At least 7 genes of the left arm (from l(3)80Fd to l(3)80Fj) map to segment h49-h51, while the most distal genes (from l(3)80Fa to l(3)80Fc) lie within the h47-h49 portion. The two right arm essential genes, l(3)81Fa and l(3)81Fb, are both located within the distal h58 segment. Intriguingly, a major part of chromosome 3 heterochromatin was found to be "empty," in that it did not contain either known genes or known satellite DNAs.     

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)     

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.     

The Heterochromatin-Associated Protein Hp-1 Is an Essential Protein in Drosophila with Dosage-Dependent Effects on Position-Effect Variegation

Chromosome rearrangements which place euchromatic genes adjacent to a heterochromatic breakpoint frequently result in gene repression (position-effect variegation). This repression is thought to reflect the spreading of a heterochromatic structure into neighboring euchromatin. Two allelic dominant suppressors of position-effect variegation were found to contain mutations within the gene encoding the heterochromatin-specific chromosomal protein HP-1. The site of mutation for each allele is given: one converts Lys169 into a nonsense (ochre) codon, while the other is a frameshift after Ser10. In flies heterozygous for one of the mutant alleles (Su(var)2-504), a truncated HP-1 protein was detectable by Western blot analysis. An HP-1 minigene, consisting of HP-1 cDNA under the control of an Hsp70 heat-inducible promoter, was transduced into flies by P element-mediated germ line transformation. Heat-shock driven expression of this minigene results in elevated HP-1 protein level and enhancement of position-effect variegation. Levels of variegating gene expression thus appear to depend upon the level of expression of a heterochromatin-specific protein. The implications of these observations for mechanism of heterochromatic position effects and heterochromatin function are discussed.     

Developmental timing and tissue specificity of heterochromatin-mediated silencing.

Heterochromatic position-effect variegation (PEV) describes the mosaic phenotype of a euchromatic gene placed next to heterochromatin. Heterochromatin-mediated silencing has been studied extensively in Drosophila, but the lack of a ubiquitous reporter gene detectable at any stage has prevented a direct developmental characterization of this phenomenon. Current models attribute variegation to the establishment of a heritable silent state in a subset of the cells and invoke differences in the timing of silencing to explain differences in the patch size of various mosaic patterns. In order to follow the course of heterochromatic silencing directly, we have generated Drosophila lines variegating for a lacZ reporter that can be induced in virtually all cells at any developmental stage. Our data indicate that silencing begins in embryogenesis and persists in both somatic and germline lineages. A heterogeneity in the extent of silencing is also revealed; silencing is suppressed in differentiated tissues but remains widespread in larval imaginal discs containing precursor cells for adult structures. Using eye development as an example, we propose that the mosaic phenotype is determined during differentiation by a variegated relaxation in heterochromatic silencing. Though unpredicted by prevailing models, this mechanism is evident in other analogous systems.     

Evidence for Intrinsic Differences in the Formation of Chromatin Domains in Drosophila Melanogaster

The results of an investigation into intrinsic differences in the formation of two different heterochromatic domains are presented. The study utilized two different position effect variegation mutants in Drosophila melanogaster for investigating the process of compacting different stretches of DNA into heterochromatin. Each stretch of DNA encodes for a gene that affects different aspects of bristle morphology. The expression of each gene is prevented when it is compacted into heterochromatin thus the genes serve as effective reporter systems to monitor the spread of heterochromatin. Both variegating mutants are scored in the same cell such that environmental and genetic background differences are unambiguously eliminated. Any differences observed in the repression of the two genes must therefore be the result of intrinsic differences in the heterochromatic compaction process for the two stretches of DNA. Studies of the effects different enhancers of variegation have upon the compaction of the two genes indicate each compaction event occurs independently of the other, and that different components are involved in the two processes. These results are discussed with regard to spreading heterochromatin and the role this process may play in regulating gene expression.     

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.     

Nuclear structure and gene activity in human differentiated cells

The nuclear arrangement of the ABL, c-MYC, and RB1 genes was quantitatively investigated in human undifferentiated HL-60 cells and in a terminally differentiated population of human granulocytes. The ABL gene was expressed in both cell types, the c-MYC gene was active in HL-60 cells and down-regulated in granulocytes, and expression of the RB1 gene was undetectable in HL-60 cells but up-regulated in granulocytes. The distances of these genes to the nuclear center (membrane), to the center of the corresponding chromosome territory, and to the nearest centromere were determined. During granulopoesis, the majority of selected genetic structures were repositioned closer to the nuclear periphery. The nuclear reposition of the genes studied did not correlate with the changes of their expression. In both cell types, the c-MYC and RB1 genes were located at the periphery of the chromosome territories regardless of their activity. The centromeres of chromosomes 8 and 13 were always positioned more centrally within the chromosome territory than the studied genes. Close spatial proximity of the c-MYC and RB1 genes with centromeric heterochromatin, forming the chromocenters, correlated with gene activity, although the nearest chromocenter of the silenced RB1 gene did not involve centromeric heterochromatin of chromosome 13 where the given gene is localized. In addition, the role of heterochromatin in gene silencing was studied in retinoblastoma cells. In these differentiated tumor cells, one copy of the RB1 gene was positioned near the heterochromatic chromosome X, and reduced RB1 gene activity was observed. In the experiments presented here, we provide evidence that the regulation of gene activity during important cellular processes such as differentiation or carcinogenesis may be realized through heterochromatin-mediated gene silencing.     

Transgene Repeat Arrays Interact with Distant Heterochromatin and Cause Silencing in Cis and Trans

Tandem repeats of Drosophila transgenes can cause heterochromatic variegation for transgene expression in a copy-number and orientation-dependent manner. Here, we demonstrate different ways in which these transgene repeat arrays interact with other sequences at a distance, displaying properties identical to those of a naturally occurring block of interstitial heterochromatin. Arrays consisting of tandemly repeated white transgenes are strongly affected by proximity to constitutive heterochromatin. Moving an array closer to heterochromatin enhanced variegation, and enhancement was reverted by recombination of the array onto a normal sequence chromosome. Rearrangements that lack the array enhanced variegation of white on a homologue bearing the array. Therefore, silencing of white genes within a repeat array depends on its distance from heterochromatin of the same chromosome or of its paired homologue. In addition, white transgene arrays cause variegation of a nearby gene in cis, a hallmark of classical position-effect variegation. Such spreading of heterochromatic silencing correlates with array size. Finally, white transgene arrays cause pairing-dependent silencing of a non-variegating white insertion at the homologous position.