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.     

Effect of the Suppressor of Underreplication (SuUR) gene on position-effect variegation silencing in Drosophila melanogaster

It has been previously shown that the SuUR gene encodes a protein located in intercalary and pericentromeric heterochromatin in Drosophila melanogaster polytene chromosomes. The SuUR mutation suppresses the formation of ectopic contacts and DNA underreplication in polytene chromosomes; SuUR+ in extra doses enhances the expression of these characters. This study demonstrates that heterochromatin-dependent PEV silencing is also influenced by SuUR. The SuUR protein localizes to chromosome regions compacted as a result of PEV; the SuUR mutation suppresses DNA underreplication arising in regions of polytene chromosomes undergoing PEV. The SuUR mutation also suppresses variegation of both adult morphological characters and chromatin compaction observed in rearranged chromosomes. In contrast, SuUR+ in extra doses and its overexpression enhance variegation. Thus, SuUR affects PEV silencing in a dose-dependent manner. However, its effect is expressed weaker than that of the strong modifier Su(var)2-5.     

Characterization of sequences associated with position-effect variegation at pericentric sites in Drosophila heterochromatin

In a variety of organisms, euchromatic genes brought into juxtaposition with pericentric heterochromatin show position-effect variegation (PEV), a silencing of gene expression in a subset of the cells in which the gene is normally expressed. Previously, a P-element mobilization screen identified transgenic Drosophila stocks showing PEV of an hsp70-white ⁺ reporter gene; transgenes in many of these stocks map to the chromocenter of polytene chromosome. A screen at an elevated temperature identified two stocks that under standard culture temperatures show complete repression of the hsp70-white ⁺ transgene. The transgenes in both cases map to the chromocenter of polytene chromosomes. Different types of middle repetitive elements are adjacent to seven pericentric transgenes; unique sequences are adjacent to two of the perimetric transgenes. All of the transgenes show suppression of PEV in response to a mutation in the gene encoding heterochromatin protein 1 (HP1). This suppression correlates with a more accessible chromatin structure. The results indicate that a pericentric transgene showing PEV can be associated with different types of DNA sequences, while maintaining a common association with the chromosomal protein HP1. Received: 15 January 1998; in revised form: 27 May 1998 / Accepted: 4 September 1998     

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.     

DNA representation of variegating heterochromatic P-element inserts in diploid and polytene tissues ofDrosophila melanogaster

Position-effect variegation (PEV) is the mosaic expression of a euchromatic gene brought into juxtaposition with heterochromatin. Fourteen different transformedDrosophila melanogaster lines with variegating P-element inserts were used to examine the DNA levels of these transgenes. Insert sites include pericentric, telomeric and fourth chromosome regions. Southern blot analyses showed that the heterochromatichsp26 transgenes are underrepresented 1.3- to 33-fold in polytene tissue relative to the endogenous euchromatichsp26 gene. In contrast, the heterochromatichsp26 transgenes are present in approximately the same copy number as the endogenous euchromatichsp26 gene in diploid tissue. It appears unlikely that DNA loss could account for the lack of gene expression in diploid tissues seen with these examples of PEV.     

l(1)pole hole is required maternally for pattern formation in the terminal regions of the embryo

Maternal expression of the l(1)pole hole (l(1)ph) gene product is required for the development of the Drosophila embryo. When maternal l(1)ph+ activity is absent, alterations in the embryonic fate map occur as visualized by the expression of segmentation genes fushitarazu and engrailed. If both maternal and zygotic activity is absent, embryos degenerate around 7 h of development. If only maternal activity is missing, embryos complete embryogenesis and show deletions of both anterior and posterior structures. Anteriorly, structures originating from labral and acron head regions are missing. Posteriorly, abdominal segments A8, 9 and 10, the telson and the proctodeum are missing. Similar pattern deletions are observed in embryos derived from the terminal class of female sterile mutations. Thus, the maternal l(1)ph+ gene product is required for the establishment of cell identities at the anterior and posterior poles of the Drosophila embryo.     

The maternal effect of lethal(1)discs-large-1: a recessive oncogene of Drosophila melanogaster

The maternal effect phenotypes of recessive mutations at the Drosophila zygotic lethal gene l(1)discs-large-1 (l(1)dlg-1) are described. L(1)dlg-1 is located in 10B7-8 on the salivary gland chromosome map. A complex complementation pattern is observed among the nine characterized alleles. Larvae missing zygotic l(1)dlg-1+ gene activity die due to aberrant growth of imaginal cells at the larval-pupal transition. Embryos lacking both maternal and zygotic activity of l(1)dlg-1+, i.e., embryos derived from homozygous l(1)dlg-1 germ line clones for null alleles, show neurogenesis and morphogenesis defects that result in very abnormal embryos. Although differentiated, most tissues are morphologically misshapen. This maternal effect is rescuable to some extent. One allele, l(1)dlg-1HF321, is a temperature-sensitive mutation for the zygotic lethality. Embryos derived from homozygous l(1)dlg-1HF321 females at 18 degrees C exhibit defects associated with dorsal closure and head involution. More extreme phenotypes are observed when females are shifted to higher temperatures and include defective dorsal closure, collapse of the somatic musculature, and an oversized central nervous system. The possible involvement of the recessive oncogene l(1)dlg-1 in cell adhesion is discussed.     

The Y-Chromosomal and Autosomal Male-Determining M Factors of Musca Domestica Are Equivalent

In Musca domestica, male sex is determined by a dominant factor, M, located either on the Y, the X or on an autosome. M prevents the activity of the female-determining gene F. In the absence of M, F becomes active and dictates female development. The various M factors may represent translocated copies of an ancestral Y-chromosomal M. Double mutants and germ line chimeras show that M(Y), M(I), M(II), M(III) and M(V) perform equivalent functions. When brought into the female germ line, they predetermine male development of the offspring. This maternal effect is overruled by the dominant female-determining factor F(D). M(I) and M(II) are weak M factors, as demonstrated by the presence of yolk proteins in M(I)/+ males and by the occurrence of some intersexes among the offspring that developed from transplanted M(I)/+ and M(II)/+ pole cells. The arrhenogenic mutation Ag has its focus in the female germ line and its temperature-sensitive period during oogenesis. We propose that M(I) and Ag represent allelic M factors that are affected in their expression. Analysis of mosaic gonads showed that in M. domestica the sex of the germ line is determined by inductive signals from the surrounding soma. We present a model to account for the observed phenomena.     

The Ketel(D) dominant-negative mutations identify maternal function of the Drosophila importin-beta gene required for cleavage nuclei formation

The Ketel(D) dominant female-sterile mutations and their ketel(r) revertant alleles identify the Ketel gene, which encodes the importin-beta (karyopherin-beta) homologue of Drosophila melanogaster. Embryogenesis does not commence in the Ketel(D) eggs deposited by the Ketel(D)/+ females due to failure of cleavage nuclei formation. When injected into wild-type cleavage embryos, cytoplasm of the Ketel(D) eggs does not inhibit nuclear protein import but prevents cleavage nuclei formation following mitosis. The Ketel(+) transgenes slightly reduce effects of the Ketel(D) mutations. The paternally derived Ketel(D) alleles act as recessive zygotic lethal mutations: the Ketel(D)/- hemizygotes, like the ketel(r)/ketel(r) and the ketel(r)/- zygotes, perish during second larval instar. The Ketel maternal dowry supports their short life. The Ketel(D)-related defects originate most likely following association of the Ketel(D)-encoded mutant molecules with a maternally provided partner. As in the Ketel(D) eggs, embryogenesis does not commence in eggs of germline chimeras with ketel(r)/- germline cells and normal soma, underlining the dominant-negative nature of the Ketel(D) mutations. The ketel(r) homozygous clones are fully viable in the follicle epithelium in wings and tergites. The Ketel gene is not expressed in most larval tissues, as revealed by the expression pattern of a Ketel promoter-lacZ reporter gene.     

Modifiers of terminal deficiency-associated position effect variegation in Drosophila

Terminal deletions of a Drosophila minichromosome (Dp(1;f)1187) dramatically increase the position effect variegation (PEV) of a yellow(+) body-color gene located in cis. Such terminal deficiency-associated PEV (TDA-PEV) can be suppressed by the presence of a second minichromosome, a phenomenon termed "trans-suppression." We performed a screen for mutations that modify TDA-PEV and trans-suppression. Seventy suppressors and enhancers of TDA-PEV were identified, but no modifiers of trans-suppression were recovered. Secondary analyses of the effects of these mutations on different PEV types identified 10 mutations that modify only TDA-PEV and 6 mutations that modify TDA-PEV and only one other type of PEV. One mutation, a new allele of Su(var)3-9, affects all forms of PEV, including silencing associated with the insertion of a transgene into telomeric regions (TPE). This Su(var)3-9 allele is the first modifier of PEV to affect TPE and provides a unique link between different types of gene silencing in Drosophila. The remaining mutations affected multiple PEV types, indicating that general PEV modifiers impact TDA-PEV. Modifiers of TDA-PEV may identify proteins that play important roles in general heterochromatin biology, including proteins involved in telomere structure and function and the organization of chromosomes in the interphase nucleus.     

SU(VAR)3-9 is a conserved key function in heterochromatic gene silencing

This review summarizes genetic, molecular and biochemical studies of the SU(VAR)3-9 protein and the evidence for its key role in heterochromatin formation and heterochromatic gene silencing. The Su(var)3-9 locus was first identified as a dominant modifier of position-effect variegation (PEV) in Drosophila melanogaster. Together with Su(var)2-5 and Su(var)3-7, Su(var)3-9 belongs to the group of haplo-suppressor loci which show a triplo-dependent enhancer effect. All three genes encode heterochromatin-associated proteins. Su(var)3-9 is epistatic to the PEV modifier effects of Su(var)2-5 and Su(var)3-7, and it also dominates the effect of the Y chromosome on PEV. These genetic data support a central role of the SU(VAR)3-9 protein in heterochromatic gene silencing, one that is correlated with its activity as a histone H3-K9 methyltransferase (HMTase). In fact, SU(VAR)3-9 is the main chromocenter-specific HMTase of Drosophila. SU(VAR)3-9 and HP1, the product of Su(var)2-5, are main constituents of heterochromatin protein complexes and the interaction between these two proteins is interdependent. Functional analysis in fission yeast, Drosophila and mammals demonstrate that SU(VAR)3-9-dependent gene silencing processes are conserved in these organisms. This is also demonstrated by the rescue of Drosophila Su(var)3-9 mutant phenotypes with human SUV39H1 transgenes.     

Multiple functions of a Drosophila homeotic gene, zeste-white 3, during segmentation and neurogenesis

Lack of both maternal and zygotic gene activity at the zeste-white 3 (zw3) locus causes severe developmental transformations. Embryos derived from germ cells that lack zw3+ gene activity die during embryogenesis and have a phenotype that is similar to that of embryos mutant for the segment polarity gene naked (nkd). In both nkd and germ line clone-derived zw3 embryos the pattern elements derived from the anterior-most part of each segment, the denticle belts, are deleted. Similar abnormal patterns of the zygotically expressed genes engrailed and Ultrabithorax are detected in both mutants, suggesting that the two genes are involved in the same developmental process. Additionally, the induction of clones of zw3 mutant cells in imaginal discs causes homeotic transformations of noninnervated hair cells into innervated sensory bristles. The multiple roles of zw3 during development and its possible interactions with the zygotic gene nkd are discussed.     

E(var)3-9 of Drosophila melanogaster encodes a zinc finger protein

The importance of a gene's natural chromatin environment for its normal expression is poignantly illustrated when a change in chromosome position results in variable gene repression, such as is observed in position effect variegation (PEV) when the Drosophila melanogaster white (omega) gene is juxtaposed with heterochromatin. The Enhancer of variegation 3-9 [E(var)3-9] gene was one of over a hundred loci identified in screens for mutations that dominantly modify PEV. Haploinsufficiency for E(var)3-9 enhances omegam4 variegation, as would be expected from increased heterochromatin formation. To clarify the role of E(var)3-9 in chromosome structure, the gene has been cloned and its mutant alleles characterized. The involvement of E(var)3-9 in structure determination was supported by its reciprocal effects on euchromatic and heterochromatic PEV; E(var)3-9 mutations increased expression of a variegating heterochromatic gene in two tissue types. E(var)3-9 mutations also had a recessive phenotype, maternal effect lethality, which implicated E(var)3-9 function in an essential process during embryogenesis. Both phenotypes of E(var)3-9 mutations were consistent with its proposed function in promoting normal chromosome structure. The cloning of E(var)3-9 by classical genetic methods revealed that it encodes a protein with multiple zinc fingers, but otherwise novel sequence.     

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.     

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.     

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.