Copyright © 1998 by the Genetics Society of America Comparative Analysis of Position–Effect Variegation Mutations in Drosophila melanogaster Delineates the Targets of Modifiers Georgette L. Sass and Steven Henikoff Howard Hughes Medical Institute, Fred Hutchinson Cancer Research Center, Seattle, Washington 98109 Manuscript received August 1, 1997 Accepted for publication October 10, 1997 ABSTRACT 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 in- clude the inversions In(1)wm4 and In(2R)bwVDe2, which are classical chromosomal rearrangements that typify PEV mutations. The other mutations are a derivative of brownDominant, in which brown1 reporters are inacti- vated by a large block of heterochromatin, and a P[white1] transposon insertion associated with second chromosome heterochromatin. In general, we find that class I modifiers affect both classical and nonclassi- cal 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 ob- servations support a model in which there are different constraints on the process of heterochromatin- induced silencing in classical vs. nonclassical PEV mutations. ENE expression depends on both intrinsic regula- presumed to reflect an intrinsic difference between G tory mechanisms, including enhancer–promoter heterochromatin and euchromatin with respect to interactions, and chromosomal context, including chro- chromatin structure and gene expression. matin structure. Whereas intrinsic regulatory mech- Genic modifiers of PEV have been readily recovered anisms are well defined molecularly, chromosomal con- in different screens (Dorn et al. 1993b; Locke et al. text is difficult to assess and is sometimes revealed only 1988; Sinclair et al. 1989, 1992; Wustmann et al. 1989) by gene-silencing phenomena. Examples of gene si- and have been considered a valuable tool in the study lencing include X-chromosome inactivation and paren- of heterochromatin. These modifiers have been catego- tal imprinting in mammals, telomere and mating-type rized into two classes (Locke et al. 1988). Class I modifi- silencing in yeast, as well as heterochromatin-induced ers act to suppress PEV when only one dose of the wild- gene silencing known as position–effect variegation type gene is present and may enhance variegation in (PEV) in Drosophila melanogaster (for review see Hen- three doses. In genetic screens, class I modifiers were drich and Willard 1995). In PEV, chromosomal rear- recovered as Suppressors of variegation [Su(var)s], which rangements that change the position of a gene so that are gene mutations or deficiencies, and as chromosomal it is placed near heterochromatin result in the variable duplications that acted as Enhancers of variegation [E(var)s]. expression of the gene. In contrast to gene-rich euchro- Because suppression of a PEV phenotype indicates a matin, heterochromatin has comparatively few genes, reduction in the ability of heterochromatin to silence a remains condensed throughout the cell cycle, and is gene, Su(var)s were predicted to be mutations in genes enriched in satellite and middle repetitive DNA se- that code for either structural components of hetero- quences (reviewed in Csink et al. 1997). Although gene chromatin or proteins that regulate heterochromatin inactivation is a direct consequence of relocation to components (Locke et al. 1988). Class II modifiers, iso- heterochromatin, it is not a normal function of hetero- lated as mutations in E(var) genes, enhance variegation chromatin, as evidenced by the presence of expressed in one dose and may suppress in three doses. Therefore, genes in heterochromatin (Gatti and Pimpinelli E(var) genes might code for proteins that antagonize the 1992). Nevertheless, the ability to inactivate a gene is silencing potential of heterochromatin or promote the formation of euchromatin (Locke et al. 1988). More than 100 dosage-dependent modifiers of PEV Corresponding author: Steven Henikoff, Fred Hutchinson Cancer Locke Reuter Research Center, A1-162, 1100 Fairview Ave. N., P.O. Box 19024, Seat- have been identified ( et al. 1988; and tle, WA 98109-1024. E-mail: [email protected] Spierer 1992; Wustmann et al. 1989). Most of these Genetics 148: 733–741 ( February, 1998) 734 G. L. Sass and S. Henikoff were identified by phenotypic suppression or enhance- (Trl ), which codes for the GAGA protein (Farkas et al. ment of the chromosomal rearrangement In(1)wm4 1994), would modify the phenotype of bwD, but other (wm4). This inversion positions the white1 (w1) gene modifiers would not. To differentiate between these within 25 kb of pericentric heterochromatin, and the and other possibilities, we examined a collection of resulting mosaic inactivation of w1 produces patches of PEV modifiers for effects on a panel of both classical white tissue in a normally red eye (Tartof et al. 1984). and nonclassical PEV mutations. Our results indicate The enhancement or suppression of this phenotype that insensitivity to class II modifiers is not restricted to can be readily scored as more or less white tissue, re- the bwD mutation, but extends to two nonclassical PEV spectively, in a genetic screen. Modifiers of PEV iso- mutations, a derivative of bwD and a variegating w1 lated using wm4 have been tested with other PEV transgene insertion into heterochromatin. We rational- mutations, such as the chromosomal rearrangements ize these findings in terms of differences in targets of In(2R)bwVDe2, In(1)y3p, and T(2:3)SbV which affect the action between class I and class II modifiers. Addition- brown, yellow, and Stubble genes, respectively. Modifiers ally, our observations point to potentially different to- isolated using wm4 typically behave similarly with these pological causes of heterochromatin-induced silencing other PEV mutations. in classical vs. nonclassical PEV mutations. A different response to genic modifiers was reported for PEV associated with the brownDominant (bwD) mutation. MATERIALS AND METHODS bwD contains a large heterochromatic insertion into the coding region of the brown1 (bw1) gene, generating a Fly stocks: Flies were reared on standard cornmeal-molas- null allele (Henikoff et al. 1993). The heterochroma- ses medium in vials or bottles at a constant temperature of 8 tin of the bwD insertion is able to silence a wild-type 25 . Four PEV mutations were used in these experiments (Fig- 1 ure 1). The isolation and characterization of the Byron mu- copy of the bw gene that is present on the homolog. tation has been described (Henikoff et al. 1995). This is referred to as trans-inactivation, and such domi- P[w1tARry1t7.2AR 5 wAR]B133-0923 (referred to in this paper as nant PEV is characteristic of all variegating alleles of P[wAR]B133) is a w1 transgene insertion in or near chromo- brown. A genetic screen designed to recover dominant some 2R heterochromatin from R. Levis, Syracuse University, modifiers of bwD identified unlinked mutations that NY. The collection of genic modifiers of PEV used in this study include both molecularly characterized mutations (ref- suppressed trans-inactivation but failed to detect com- parable enhancer mutations (Talbert et al. 1994). A collection of these suppressors of bwD were tested for their effect on wm4, and 33 out of 37 were found to be typical class I modifiers [i.e., Su(var) mutations]. The notable absence of enhancers of bwD was not caused by an inability to detect enhancement of the phenotype because two dominant enhancer mutations, both in- volving a rearrangement of the bwD chromosome, were identified. In addition, the bwD mutation is enhanced in males that lack a Y chromosome (P. Talbert, per- sonal communication), indicating that bwD responds to this modifier in a manner that is consistent with other PEV mutations (Spofford 1976). These observations suggest that classical PEV mutations (i.e., gross chromo- somal rearrangements such as wm4) and nonclassical PEV mutations such as bwD exhibit differential re- sponses to PEV modifiers, specifically to class II modi- fiers. Failure to recover class II modifiers (i.e., E(var) muta- tions) in the collection of modifiers of bwD could be ex- Figure 1.—Depictions of PEV mutations used for compar- plained if the linkage enhancers of bwD were exception- ative analysis. The classical PEV mutation wm4 is a chromo- 1 ally strong, and therefore weaker effects would have somal inversion in which the w gene is moved proximally to within 25 kb of X chromosome heterochromatin. In the case been undetected. Alternatively, the lack of enhancers of bwVDe2, the bw1 gene is moved close to 2R heterochromatin. D may be indicative of a bw -specific property. For exam- In each case, the small arrows show the position of the inver- ple, the bwD heterochromatic insertion is composed sion breakpoints. The nonclassical PEV mutation Byron is a primarily of the simple sequence satellite (AAGAG) duplication of the 59E region in which the proximal element n D D (Csink and Henikoff 1996), which is present in het- carries bw and the distal element carries a wild-type bw gene. P[wAR]B133 is a nonclassical PEV mutation in which a erochromatin (Lohe et al. 1993), and bound by GAGA 1 Raff w transgene has inserted into or very near 2R heterochroma- protein in early embryos ( et al. 1994). This raises tin. For PEV mutations involving the second chromosome, the possibility that the class II modifier Trithorax-like only the right arm is depicted. Targets of Variegation Modifiers 735 TABLE 1 Molecularly characterized modifiers of PEV Modifier Protein product Reference Class I Su(var)2-5 Heterochromatin protein 1, HP1, associates with Eissenberg et al.