Vig and vig2 mutations are modifiers of variegation The PEV system provides a very sensitive assay for the involvement of candidate genes in heterochromatin formation. In this study we used yellow variegating reporters located in regions 57h (B79) and 49h (J545) of chromosome 3, and within centric region 10h of chromosome Y (J448) [32,33] to evaluate the ability of vig and vig2 mutations to affect PEV. The vigEP812 mutation is caused by a P{EP} insertion within the vig coding region (Figure S3A) that results in severe reduction of the VIG protein (Figure 3A). PBac{GAL4D,EYFP} resides in the vig2 gene [34] and disrupts the production of Vig2 protein (Figure 3B). We generated flies carrying the yellow PEV reporters and mutations in either vig or vig2: y1w67c23/Y; vigEP812/+; P(y+)B79/+ y1w67c23/Y; +; P(y+)B79/vig2PL470 y1w67c23/Y; vigEP812/+; P(y+)J545/+ y1w67c23/Y; +; P(y+)J545/vig2PL470 y1w67c23/Y, P(y+)J448; vigEP812/+; + y1w67c23/Y, P(y+)J448; +; vig2PL470/+. To score the variegating yellow phenotype we prepared male abdominal cuticles and compared the number of dark patches to the WT control. The control flies were obtained simultaneously in a separate cross between males carrying the reporter and y1w67c23 females (for example: y1w67c23/Y; +; P(y+)B79/+). (This control cross is necessary because it is inappropriate to use siblings carrying the PEV reporter and a balancer chromosome as a WT control, as the balancer chromosomes often carry modifiers of PEV). However, the vigEP812 mutation affects the vasa gene as well as vig (Figure S3A), and any suppression of PEV in this case could be attributed to the deficit in Vasa protein. To resolve this issue we crossed a stock carrying the yellow PEV reporter P(y+)B79/+ and the vasaAS mutant line (Figure S3A), in which only the vasa gene is affected [35] and vig expression is at wild type level (Figure S3B). Analysis of the progeny phenotype demonstrated that the vasaAS mutation does not modify PEV (Figure S3C), indicating that the suppression observed with the vigEP812 mutation is caused by a reduction in the levels of VIG. Silencing of the yellow reporter B79 was relieved in a vigEP812 or vig2PL470 heterozygote mutant background (Figure 3C, panels 1– 3). The J545, reporter located in a different heterochromatic domain, was not sensitive to the vigEP812 mutation; but vig2PL470 resulted in enhancement of variegation (Figure 3C, panels 4–6). The J448 P-element is inserted in the centric region of the Y chromosome; flanking sequences include 1360 and Su(Ste). Both vigEP812 or vig2PL470 mutations caused suppression of variegation at this location (Figure 3C, panels 7–9). The differences seen with various PEV reporters are not without precedent; Su(var)3-9 mutations result in suppression of variegation for reporters in the pericentric heterochromatin, but enhancement of variegation for reporters in the fourth chromosome [36,37]. Overall, the results of the yellow PEV tests suggest an involvement of VIG and Vig2 proteins in heterochromatin formation or maintenance. However, the PEV reporters’ readouts indicate that VIG and Vig2, unlike HP1, are not heterochromatin ‘construction material’; our data suggest that vig2 gene is rather involved in targeting (nucleation) of heterochromatic domains. Mutations in this gene may cause the disruption of proper distribution of heterochromatin structural components; as a result we observe suppression or enhancement of variegation. One can speculate that Vig2 is epistatic to VIG in this process. Vig2 is prominent in the early embryo, when heterochromatin formation is initiated, while VIG is expressed at higher levels in larval stages, when heterochomatin formation must be maintained. VIG and Vig2 Impact Chromatin VIG and Vig2 partially overlap with HP1 on polytene chromosomes Large polytene chromosomes are found in the larval salivary glands of Drosophila. Immunostaining of these chromosomes with antibodies allows detection and analysis of the distribution pattern of any nuclear protein in vivo. HP1 stains predominantly the chromocenter, telomeres, and the small 4th chromosome, the domains where most of the heterochromatin is found. In addition, HP1 also marks numerous sites on the chromosome arms, including the ‘goose neck’, a cluster of bands on the 2nd chromosome [38], and large puffs (sites of intense transcription) [39]. To visualize the distribution of VIG and Vig2 relatively to HP1 we treated WT polytene chromosomes with antibodies generated against the C-terminus of VIG (CHS1803) and a mouse monoclonal antibody specific for HP1 (C1A9) (Figure 4A). CHS1803 recognizes both VIG and Vig2; it was used in preference to other VIG antibodies because of its high sensitivity and the absence of cross- reacting bands on a Western blot using total protein from salivary glands (Figure S4). The CSH1803 antiserum stains the chromo- center and produces a complex pattern on the chromosome arms. Figure 4A shows the overlap between HP1 signal and the signal produced by CSH1803 at the chromocenter (arrow 1) and at some sites on chromosome arms. Only partial overlap is observed; for example, the ‘goose neck’ region, prominently associated with HP1, is devoid of both VIG and Vig2 (arrow 3). Among numerous sites on the chromosomal arms marked by CSH1803, only some are shared with HP1 (arrow 2). To obtain the individual distribution patterns of VIG and Vig2, we used salivary glands from homozygous vigEP812 or vig2PL470 mutant larvae, after having verified by immunoblot (Figure 4B) that in the absence of VIG only Vig2 is recognized by this antibody and vice versa. The distribution pattern of Vig2 seen on vigEP812 polytene chromosomes (Figure 4C) clearly shows overlap between HP1 and Vig2 at the chromocenter and at some sites on the chromosome arms, including sites of active transcription (large puffs). By contrast, VIG staining of vig2PL470 chromosomes presents different features (Figure 4D). There is little signal present at the chromocenter and intense staining is seen at scattered sites on the chromosome arms, including some large puffs where HP1 is also present. Thus, VIG localization on polytene chromosomes differs from that of Vig2. In particular, Vig2 association with constitutive heterochromatin domains suggests that this protein could be involved in heterochromatin formation and/or maintenance. On the other hand, VIG overlaps with HP1 primarily at some sites on the chromosome arms, including puffs, suggesting either a direct role in heterochromatin formation or participation in regulatory processes that have indirect effects on heterochromatin. The H3K9me2 level is decreased in the vig2PL470 mutant, but not in vigEP812 To further investigate the role that VIG and Vig2 play in the process of heterochromatin formation, we looked at the hallmark of silent chromatin - H3K9 dimethylation. We prepared protein extracts from adult flies and assessed the level of H3K9me2 by quantitative Western blot (Figure 5). For reference we used the H3K9me2 levels of the Oregon R (WT) strain and the Su(var)3-906 mutant flies. The Su(var)3-9 gene encodes one of three known histone H3K9 methyltransefases in Drosophila, and is an effective suppressor of PEV. Su(var)3-906 is a allele in which H3K9me2 levels are significantly decreased in embryos [40]. In adult flies the dimethylation of H3K9 in a homozygous Su(var)3-9 mutant background fell by ca. 50% relatively to WT. The homozygous vigEP812 mutant did not show any loss of H3K9 methylation, but the homozygous vig2PL470 mutant clearly demonstrated a decrease PLoS ONE | www.plosone.org 4 July 2009 | Volume 4 | Issue 7 | e6182