VIG and Vig2 Impact Chromatin Figure 4. Immunofluorescent staining of polytene chromosomes. (A) VIG and Vig2 (visualized using CSH1803) show a distribution pattern that overlaps with HP1 at the chromocenter (arrow 1) and in some cases on the chromosome arms (arrow 2) of the WT polytene chromosomes. However, the majority of the cytobands do not overlap; for instance, region 31 on the 2nd chromosome (the ‘goose neck’) is associated with HP1 alone (arrow 3). The lower row of images shows staining at the chromocenter. (B) Western blot performed using protein from salivary glands shows detection of both VIG and Vig2 protein by the CSH1803 antibody. In the vigEP812 mutant only Vig2 is present; in the cg11844PL470 mutant only VIG is recognized. (C) Polytene chromosomes were obtained from vigEP812 mutant larvae; in the absence of VIG we observe significant overlap between HP1 and Vig2 protein at the chromocenter and at some sites on the chromosome arms. The lower row of images shows staining at the chromocenter. (D) The distribution of VIG seen on vig2PL470 mutant polytene chromosomes shows multiple overlapping sites shared by HP1 and VIG on the chromosomal arms, but only faint speckles of VIG at the chromocenter. The lower row of images shows staining at the chromocenter. doi:10.1371/journal.pone.0006182.g004 to ca. 60% of WT levels. We also checked different types of tissues. Adult heads (soma) and ovaries (germline) both exhibit the same pattern of H3K9me2 depletion as seen in whole adult flies (Figure S5). In conclusion, while both vig and vig2 mutants can be dominant suppressors of PEV, these experiments show that vig2 impacts H3K9 methylation, a key signature of heterochromatin, while vig does not. HP1 and HP2 (Large isoform) are depleted in the nuclei of a vig mutant Next we extended our analysis to another major component of heterochromatin, the HP1 protein. Nuclear proteins were extracted from adult tissues of the same fly strains: Oregon R (WT), vigEP812, vig2PL470, and Su(var)3-906. Because prior studies have shown that in homozygous Su(var)3-906 mutant larvae HP1 is PLoS ONE | www.plosone.org 6 July 2009 | Volume 4 | Issue 7 | e6182