Comparative Analysis of Position–Effect Variegation Mutations in Drosophila Melanogaster Delineates the Targets of Modifiers

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 include 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 brownϩ reporters are inactivated by a large block of heterochromatin, and a P[whiteϩ] 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.

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 whiteϩ (wϩ) 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 wϩ 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 wϩ 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 brownϩ (bwϩ) 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 Њ tin of the bwD insertion is able to silence a wild-type 25 . Four PEV mutations were used in these experiments (Fig- ϩ 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[wϩtARryϩt7.2AR ϭ wAR]B133-0923 (referred to in this paper as nant PEV is characteristic of all variegating alleles of P[wAR]B133) is a wϩ 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 involving a rearrangement of the bwD chromosome, were identified. In addition, the bwD mutation is enhanced in males that lack a Y chromosome (P. Talbert, personal 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 chromosomal rearrangements such as wm4) and nonclassical PEV mutations such as bwD exhibit differential responses to PEV modifiers, specifically to class II modifiers. Failure to recover class II modifiers (i.e., E(var) mutations) 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- ϩ 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 bwϩ 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 ϩ 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. 1990 heterochromatin and contains a chromodomain Su(var)3-6 Protein phosphatase 1, catalytic subunit Baksa et al. 1993 Su(var)3-7 Protein contains seven widely spaced zinc ﬁngers Cleard et al. 1995 Su(var)3-9 Protein contains chromodomain and trx homology Tschiersch et al. 1994 (SET domain) Class II E(var)3-93D Protein contains BTB domain and associates with Dorn et al. 1993a; polytene chromosomes Zollman et al. 1994 E2F Transcriptional activator, involved in cell cycle regulation Seum et al. 1996 Trl GAGA transcriptional activator Farkas et al. 1994

erences listed in Table 1) and uncharacterized modifiers that zygous for the modifier and the marked chromosome were have been described previously (Hearn et al. 1991; Sinclair then crossed to females carrying the PEV mutation. Siblings et al. 1983) or isolated in previously published genetic screens carrying the PEV mutation with either the modifier or the (Dorn et al. 1993b). EC stocks are documented (http://fly- marked chromosome were then compared directly. For PEV base.bio.indiana.edu:82/stocks/labs/reuter2.csv). All other mutations that involved wϩ, only male siblings were compared. chromosomes or mutations are described in Lindsley and Analysis of modifier effects on PEV mutations: Pigment as- Zimm (1992). says, which are commonly used to measure the degree of bwϩ Experimental design: We examined the effects of modifi- or wϩ reporter expression, can be inaccurate because varia- ers on both classical and nonclassical PEV mutations. wm4 and tion in head size will affect pigment measurements even when bwVDe2 are classical PEV mutations in which two different re- the observed degree of variegation is similar. In addition, pig- porter genes (wϩ and bwϩ, respectively) are variably silenced ment assays lack the required sensitivity for PEV mutations because of their position next to heterochromatin (Figure 1). that result in very weak or strong inactivation. However, side- We also tested two nonclassical PEV mutations that involve by-side visual comparisons of flies can be reliable and sensitive the same reporters but are not gross chromosomal rearrange- to subtle differences. We established five phenotypic ranks for ments. In the case of Byron, a derivative of the bwDominant (bwD) each PEV mutation. Because the severity of the PEV muta- PEV mutation, the large block of heterochromatin inserted tions used in this study varies, phenotypic ranks were specific within the bwϩ gene inactivates bwϩ reporter genes on the ho- for each mutation. Also, the same predetermined phenotypic molog in trans and on a duplication. Variegating transgene in- ranks for a given PEV mutation were used when examining sertions into heterochromatin are also nonclassical PEV mu- the effect of each modifier analyzed in this study. Vials con- tations. The fourth PEV mutation we examined is a wϩ taining flies of a given genotype that had been aged 8–10 days transgene, P[wAR]B133, which is variably expressed because of at 25Њ were coded so that assignment of ranks could be made its location in or near second chromosome (2R) heterochro- without knowing the specific genotype. In cases where the rel- matin (R. Levis, personal communication). evant genotypes do not show phenotypic overlap in the distri- The modifiers used in our analysis are thought to be spe- bution of individuals falling into each rank, i.e., there is clear cific gene mutations, with the known exception being a re- suppression or enhancement despite the phenotypic varia- gional duplication [the class I E(var) Dp(2;2)E39A]. The deter- tion of individuals within a genotype, the effect of a modifier mination that a molecularly uncharacterized modifier is a can be stated unequivocally. In cases where the distributions class I or class II modifier cannot be made with certainty be- of the sibling phenotypes overlapped, however, the signifi- cause a gain-of-function mutation of one class will behave the cance of the overlap was determined statistically. The non- same as a loss-of-function mutation of the other class. For this parametric equivalent of the unpaired t-test, the Mann-Whit- reason, we concentrated primarily on those modifiers for ney U test, was used to determine significance (StatView, which the gene has been cloned and sequenced. Abacus Concepts Inc., Berkeley, CA) and P values are re- The modifier lines used in this study are either homo- ported where relevant. zygous lethal or infertile, requiring that the stocks be kept over balancer chromosomes. Previous studies assayed modifier effects by comparing the variegated phenotype with the RESULTS modifier-bearing chromosome (e.g., wm4;Modifier) to the phenotype with the balancer chromosome (e.g., wm4;Balancer). We Analysis of class I modifiers: We examined the ef- found that this was an inaccurate test of the modifier effect fects of four molecularly characterized class I modifiers because balancer chromosomes are capable of affecting the degree of variegation (data not shown). During the course of (listed in Table 1) on our collection of PEV mutations. our studies, it became clear that an internal control was As expected, all class I loss-of-function mutations sup- needed in each cross of modifier to PEV mutation. This was pressed and the class I duplication [E(var)39A] en- accomplished by first outcrossing flies carrying the modifier hanced the variegation seen in wm4 (Table 2, column 1; to a line that carried a marked chromosome. The second and 2 Figure 2, column 1). Similar results were obtained with third chromosomes were marked with the Sco and H muta- VDe2 tions, respectively. These marked chromosomes do not modify bw (Table 2, column 2; Figure 2, column 2). Class I the phenotypes of the PEV mutants used in our analysis (data modifiers likewise affect variegation of the nonclassical not shown). The resulting male progeny that were hetero- PEV mutations Byron and P[wAR]B133 (Table 2; Figure 736 G. L. Sass and S. Henikoff

TABLE 2 Effect of molecularly characterized modiﬁers

PEV mutation Modiﬁer wm4 bwVDe2 Byron P[wAR]B133 Su(var)20502 SS S S Dp(2;2)E39A E E E E (Ͻ0.0001) (Ͻ0.0001/0.0001) (0.001/0.005) (0.01) Su(var)3-6 01 SS S S Su(var)3-7 SS S S Su(var)3-901 SS X X (0.5/0.7) (0.02E) E(var)3-93D E E X X (Ͻ0.0001) (0.006/0.04) (0.1/0.2; 0.4/0.005S) (0.5) Trl⌬R85 E E X E (Ͻ0.0001) (0.001/Ͻ0.0001) (0.5/0.6; 0.6/0.9) (0.01) Trl13C E E X E (Ͻ0.0001) (0.0004/Ͻ0.0001) (Ͻ0.0001/0.6; 0.4/0.6) (0.04) Trl62 E E X X (Ͻ0.0001) (0.001/0.02) (0.06S/Ͻ0.0001S; 0.07S/0.3) (0.5) TrlR4 E E X E (Ͻ0.0001) (0.05/0.01) (1/0.7) (0.003) E2F91 E X X X (0.0006) (0.6/0.3) (1/0.4) (0.07S) For Su(var)3-7, Df(3R)Ace-HD1 was used (Reuter et al. 1987). S and E, suppression and enhancement, respectively. X, lack of expected effect. P values are shown in parentheses (male for wm4 and P[wAR]B133; male/ female for bwVDe2 and Byron).

2, columns 3 and 4, respectively). However, one class I on the Byron phenotype (Figure 2, column 3). With re- modifier, Su(var)3-9, did not suppress Byron and spect to the Trl series, there also was no enhancement P[wAR]B133 (Table 2, columns 3 and 4). A second al- of the Byron phenotype. The presence of either a puta- lele, Su(var)3-905, similarly failed to suppress these non- tive null allele of Trl (TrlR85) or the revertant allele classical PEV mutations (data not shown). (TrlR4) had no consequence for the Byron phenotype. Analysis of class II modifiers: Three class II modifi- The two P element insertion alleles of Trl (Trl13C and ers that have been molecularly characterized (listed in Trl62), considered to be hypomorphic mutations, were Table 1) were also tested with our collection of PEV also examined. The Trl62 allele did not show enhance- mutations. As previously described, wm4 was signifi- ment of the Byron phenotype. Although the distribu- cantly enhanced by all class II modifiers (Table 2, col- tion of individuals into phenotypic rankings was signifi- umn 1; Figure 2, column 1). Surprisingly, a revertant of cantly different in the presence of Trl62, it was in a the Trl13C allele weakly enhanced wm4. This revertant al- direction that indicates suppression, not enhancement. lele, TrlR4, had been generated by the precise excision Trl13C had an inconsistent effect in that males appeared of the P transposon in Trl13C and so should have no ef- to be enhanced using the rank distribution assay, fect on PEV mutants (Farkas et al. 1994). The TrlR4 whereas females were not affected. Enhancement in chromosome also enhanced the phenotype of bwVDe2 Trl13C males was too weak to be detected using pigment (see below), indicating that modification is not specific assays (data not shown). Because we can detect back- to wm4. Therefore, it appears that the genetic background enhancement of wm4 and bwVDe2 by a revertant ground of the Trl13C chromosome and derivatives TrlR4 of Trl13C, the weak enhancement attributable to this and TrlR85 contribute to the modifying effects of muta- chromosome is suspect. Thus, we conclude that class II tions in the Trl gene. modifiers do not enhance the phenotype of Byron. We also examined the classical PEV mutation bwVDe2 P[wAR]B133 was also examined with the class II mod- (Table 2, column 2). Like wm4, bwVDe2 was also enhanced ifiers (Table 2, column 4). E(var)3-93D and E2F had by the class II modifiers Trl and E(var)3-93D (Figure 2, no effect (Figure 2, column 4). The Trl62 allele had no column 2); however, the E2F mutation had no effect on significant effect either; however, the number of bwVDe2. P[wAR]B133 individuals exhibiting an enhanced pheno- The nonclassical PEV mutation Byron showed very type was increased in the presence of the null (TrlR85), different responses to class II modifiers (Table 2, col- the hypomorphic (Trl13C ), and the revertant (TrlR4 ) alle- umn 3). E(var)3-93D and E2F had no enhancing effect les. The observed enhancement of P[wAR]B133 by the Targets of Variegation Modifiers 737

Figure 2.—Phenotypes of the PEV mutants with selected modiﬁers. Shown are typical phenotypes of the four PEV mutants used in this study. Examples include the phenotype in the absence of a modiﬁer, as well as in the presence of a class I Su(var) [Su(var)3-6 in the case of wm4 and Byron; Su(var3-7) in the case of bwVDe2 and P[wAR]B133)], the class I duplication Dp(2;2)E39A, and the class II E(var)3-93D.

revertant allele TrlR4 is in keeping with the background remaining three E(var) mutations had no effect on enhancement that was detected for the classical PEV males, but they did enhance females. Five of the E(var) mutations. The fact that the independently derived mutations were also tested on P[wAR]B133 (Table 3, Trl62 allele (i.e., with a different genetic background) column 2), and four had no effect. Our analysis of mo- does not cause enhancement indicates that the non- lecularly uncharacterized modifiers extends our obser- classical PEV mutation P[wAR]B133 is not affected by vations made with the class I and class II modifiers: class Trl mutations. Thus, the class II modifiers used in this I modifiers affect all PEV mutations, whereas class II study have no effect on P[wAR]B133. modifiers affect classical PEV mutations but not non- Analysis of additional modifiers of unknown class: classical PEV mutations. A collection of 11 molecularly uncharacterized modifier mutations was used to further assess the effect of modifi- DISCUSSION ers of PEV on the nonclassical PEV mutation Byron (Table 3). Seven of these modifiers were also tested with The comparative analysis of PEV modifiers pre- P[wAR]B133. Mutations in the putative class I modifier sented here used a method that allowed detection of Su(var)2-1 result in hyperacetylation of histone H4 significant modification. Our results indicate a differ- (Dorn et al. 1986). This mutation strongly suppresses ence in susceptibility to modification between nonclas- both Byron and P[wAR]B133. Similarly, Su(var)208 strongly sical and classical PEV mutations. As demonstrated in suppresses both nonclassical PEV mutations. Of nine the studies of others, classical PEV mutations were sen- E(var) mutations tested, six were without significant ef- sitive to both class I and class II modifiers. In general, fect on the Byron phenotype (Table 3, column 1). The the nonclassical PEV mutations examined in this study 738 G. L. Sass and S. Henikoff

TABLE 3 the wϩ reporter gene in wm4, the proximity of hetero- Effect of molecularly uncharacterized modifiers chromatin may create a competition between open and closed chromatin at wϩ. Competition models for re- PEV mutation porter gene inactivation have been previously described (Aparicio and Gottschling 1994; Elgin 1996). The Modifier wm4 P[wAR]B133 Byron outcome of competition can be seen as the variegated Su(var)2-1201 SS Sphenotype. Modifiers that alter the balance between Su(var)208 SS Smaintaining an active transcriptional complex and EC7 EX Xforming heterochromatin will alter the phenotype. A EC19 ENDXcommon theme of class II modifiers appears to be the EC33 EX X potential for involvement in chromatin architecture EC66 ENDXDe Rubertis Dorn Eberl EC69 E X X/E ( et al. 1996; et al. 1993a; et al. EC70 EX X1997; Farkas et al. 1994; Seum et al. 1996). Mutations EC76 ENDXthat affect chromatin architecture would result in en- EC88 E E X/E hancement by decreasing the probability that an active EC91 E ND X/E transcriptional complex is formed at wϩ, which in turn ϩ ND indicates not determined. See Table 2 for key. Sex-specific increases the probability that w will be inactivated by differences are shown as male/female. heterochromatin. Thus, any modifier that affects the competition between maintaining an active transcriptional state and a heterochromatin-induced inactive show the expected response to class I modifiers, but state will affect the phenotype of wm4. not to class II modifiers. This was true regardless of the The same model would apply to the bwϩ reporter dosage characteristics of the modifiers tested because gene that is found in cis to the breakpoint of the inver- both haplo-dependent and haplo-, triplo-dependent sion bwVDe2. As is the case for all bw variegating muta- modifiers of a class behaved similarly. Our results con- tions, bwVDe2 is dominant and causes trans-inactivation of firm and extend inferences from previous studies that the bw gene on the wild-type homolog. Trans-inactiva- class II modifiers have no effect on nonclassical PEV tion is also sensitive to the effects of class II modifiers, mutations such as bwD (Talbert et al. 1994) or trans- as evidenced by the enhanced phenotype of bwVDe2. Be- gene insertions into heterochromatin (unpublished re- cause homologous chromosomes are paired in Dipter- sults cited in Wallrath and Elgin 1995). ans (Metz 1916), the resulting formation of an inver- Our results demonstrate that class I modifiers have a sion loop would bring the trans copy of the bwϩ general role in heterochromatin-induced silencing in reporter closer to a putative heterochromatic compart- that they affect both classical and nonclassical PEV mu- ment (Figure 3). Proximity to a heterochromatic com- tations. This is as expected if the target of class I modi- partment is responsible for the susceptibility of the fier effects is heterochromatin. In contrast, class II trans copy of the bwϩ reporter to class II modifiers. modifiers only affect classical PEV mutations. One ex- There would be an unstable balance between the forma- planation for this observation is that the insensitivity of tion of an active transcription complex on the trans copy the nonclassical PEV mutations Byron and P[wAR]B133 of bw and the influence of trans-inactivating hetero- reflects an unusual composition of heterochromatin re- chromatin. As in the case of wm4, modifiers that alter sponsible for silencing. Compositional differences have the balance between maintaining an active state and a been speculated to underlie differential responses to silenced state will affect the phenotype. modifiers among classical PEV mutations (Bishop The mechanism of reporter gene inactivation in non- 1992; Lloyd et al. 1997). However, most classical PEV classical PEV mutants may be qualitatively different than mutations respond to class II modifiers even though that for classical PEV mutants (Figure 3). In the case of these mutations represent diverse heterochromatin bwD (and its derivatives), the bwϩ reporter gene present breakpoints. Occasional failure of a classical PEV muta- on the homolog may be inactivated because it is mislo- tion to respond to a modifier does not extend to the calized to a heterochromatic compartment of the nu- class as a whole, as is the case for Byron and P[wAR]B133. cleus (Csink and Henikoff 1996; Dernburg et al. 1996; Furthermore, we do not think that the nonclassical Talbert et al. 1994). A correlation between nuclear mis- PEV mutations used in our study are unusual because localization and the degree of bw inactivation in the eye failure to respond to Trl mutations has been noted for suggests that a heterochromatic compartment is not multiple variegating transgene inserts (unpublished re- conducive to bw transcription (Csink and Henikoff sults cited in Wallrath and Elgin 1995). 1996). The effect of decreasing the dose of a gene that Alternatively, the observed differences between clas- encodes a protein involved in establishing an open sical and nonclassical PEV mutations in their response chromatin state would be negligible if reporter gene ex- to class II modifiers may reflect differences in the pression is already compromised by virtue of its mislo- mechanism of reporter gene inactivation. In the case of calization to a heterochromatic compartment. The Targets of Variegation Modifiers 739

Figure 3.—Model of PEV in classical vs. nonclassical PEV mutations. The difference in sensitivity to class II modifiers that is observed between classical and nonclassical PEV mutants in our study is caused by differences in reporter silencing. The model (see discussion for details) is il- lustrated using the classical PEV mutations bwVDe2 and the nonclassical PEV mutations bwD. In the case of bwVDe2, somatic pairing gen- erates an inversion loop that brings the bwϩ reporter of the homologous chromosome into the vicinity of pericentric heterochromatin in the inter- phase nucleus. Heterochro- matin is depicted as a wavy line. The dose of a class I or class II modifier can affect the competition between establishing an active transcriptional complex at the bwϩ reporter gene or inactivation caused by heterochromatin association. A reduction in the dose of a class I modifier decreases heterochromatic association and results in phenotypic suppression. Conversely, a reduction in the dose of a class II modifier decreases the probability of an active state and therefore increases heterochromatic association leading to an enhancement of the phenotype. In the case of the nonclassical PEV mutation bwD (or its derivative, Byron), somatic pairing of the homologs, com- bined with association between the heterochromatic insertion of bwD and second chromosome heterochromatin, results in mislo- calization of a bwϩ reporter gene to a heterochromatic compartment. The shaded oval represents a heterochromatic compartment with silencing properties. The force of heterochromatic association predominates, and therefore the dosage of class II modifiers is without phenotypic consequence. As for classical PEV mutations, heterochromatic associations in nonclassical PEV mutations are altered by changes in the dose of class I modifiers. Reducing the dose of a class I modifier decreases heterochromatic association and results in phenotypic suppression. same insensitivity would be observed for a transgene in- Su(var)3-901 was a weak suppressor of classical PEV mu- sertion in heterochromatin. In the case of P[wAR]B133, tations (data not shown). Although Su(var)3-9 was sug- the wϩ reporter is mostly inactivated (as indicated by gested to play an important role in heterochromatini- the extreme variegation) because it is sequestered zation (Tschiersch et al. 1994), our results suggest within a heterochromatic compartment, and so dosage that its primary target of action is not heterochromatin. changes in chromatin architectural proteins would not How do our results fit with other well-studied silenc- be limiting for transcription. ing phenomena? The potential mechanistic similarities There were exceptions to the above-described gen- between class I modifiers of PEV and proteins involved eralizations regarding modifier mutations. Three of the in repression of homeotic gene expression [Polycomb 12 E(var) mutations enhanced the phenotype of Byron group (Pc-G) gene products] have been discussed fre- females (but not males), and one of these also en- quently (Moehrle and Paro 1994; Pirrotta and Ras- hanced P[wAR]B133. In these cases, we predict that the telli 1994; Reuter and Spierer 1992). The class II corresponding mutation leads to a gain of function of modifiers Trl and E(var)3-93D are mutations in genes of a class I modifier gene. Alternatively, enhancement the Trithorax group, genes that encode proteins needed might be caused by a mutation in a gene that encodes for the appropriate activation of homeotic genes. This a product directly involved in the negative regulation observation led investigators to conclude that this over- of heterochromatin. Another exception, the class I lap was noteworthy (Dorn et al. 1993a; Farkas et al. modifier Su(var)3-9 did not suppress either Byron or 1994). Our results provide an explanation. If the class P[wAR]B133. This is surprising given that Su(var)3-9 was II modifier effects reflect reporter gene sensitivity, then shown to suppress associations between bwD and hetero- the loss of chromatin architectural proteins (such as chromatin in larval brains (Csink and Henikoff 1996). encoded by Trl and E(var) 3-93D) may affect susceptible Because Su(var)3-9 mutations are in a transcribed region homeotic genes by a similar mechanism. That is, loss of that is primarily expressed in embryos (Tschiersch et Trl or E(var)3-93D alters the balance of an open vs. al. 1994), the product might not be present during de- closed chromatin state, resulting in the increased prob- velopment of the eye when the bw gene is expressed. ability of the inappropriate formation of a Pc-G or a Consistent with this explanation, we found that heterochromatin-mediated silencing complex. 740 G. L. Sass and S. Henikoff

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C., 1996 Heterochromatin and gene regulation in Dro- fect gene expression. Class II modifiers act at the level sophila. Curr. Opin. Genet. Dev. 6: 193–202. of reporter gene expression; therefore, modification of Farkas, G., J. Gausz, M. Galloni, G. Reuter, H. Gyurkovics et al., gene expression is not informative with respect to the 1994 The Trithorax-like gene encodes the Drosophila GAGA factor. Nature 371: 806–808. properties of heterochromatin. If PEV is to be used as a Gatti, M., and S. Pimpinelli, 1992 Functional elements in Dro- tool in the analysis of heterochromatin, the identifica- sophila melanogaster heterochromatin. Annu. Rev. Genet. 26: 239– tion of modifiers that act on both classical and nonclas- 275. Hearn, M. G., A. Hedrick, T. A. Grigliatti and B. T. Wakimoto, sical PEV mutations would be more informative. 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