VIG and Vig2 Impact Chromatin Figure 6. Quantitative Western blots to estimate the amounts of HP1 and HP2 in adult fly nuclear preparations. (A) The vigEP812 strain shows a decrease in HP1 similar to the Su(var)3-906 mutant. In vig2PL470 flies the level of HP1 appears to be similar to WT. H4 was used as a loading control. (B) The HP2 Large isoform follows the HP1 pattern. Actin served as a loading control. Error bars represent standard error of the mean. doi:10.1371/journal.pone.0006182.g006 Figure 5. Quantitative Western blot for H3 dimethylation at Lys9. Total protein extracts were obtained from adult flies and probed with an anti-H3K9me2 specific antibody; anti-H4 was used as a loading control. A stock carrying a mutation of Su(var)3-9 histone methyltransferase was used as a control. Vig2PL470 demonstrates a diminished level of H3K9me2, whereas vigEP812 does not. Error bars represent standard error of the mean. doi:10.1371/journal.pone.0006182.g005 lost from the chromocenter of polytene chromosomes, we used this mutant line as a positive control [41]. As expected, quantitative Western blots show a loss of nuclear HP1 in Su(var)3-906 mutants. Similarly, we detected only about 50% of the WT level of HP1 in the homozygous vigEP812 mutant line, but we did not see any changes in homozygous vig2PL470 flies (Figure 6A). We considered the possibility that the vig mutation could affect the expression of Su(var)205 (most likely indirectly) and tested Su(var)205 RNA levels by RT-PCR. However, there were no changes in a comparison to the levels of Su(var)205 messenger RNA in WT and vigEP812 mutants (Figure S6). To further verify the HP1 results, we looked at a binding partner of HP1, Heterochromatin Protein 2 (HP2) [42,43]. The Su(var)2-HP2 gene codes for two HP2 protein isoforms, HP2 Large (HP2L) and HP2 Small (HP2S); HP2L binding to heterochromatin is HP1-dependent. We estimated the amount of HP2L in nuclei by quantitative Western blot (Figure 6B). The results obtained for HP2L were reflective of HP1: loss of HP2L was observed in nuclei prepared from vigEP812 and Su(var)3-906 mutant flies, but not in those from the vig2PL470 line. These results suggest that VIG might be involved in stabilizing the interaction of HP1 with chromatin, while Vig2 does not play such a role. The latter result is surprising, given that the Vig2 mutation has an effect on H3K9 methylation. HP1, VIG and Vig2 co-immunoprecipitate To examine whether VIG or Vig2 are present in a complex with HP1 we performed immunoprecipitation experiments using antibodies specific for HP1 (C1A9 and WA191), VIG (CSH1801), and Vig2 (CSH2542). In addition to co-precipitating HP2L, as expected, HP1 also co-precipitated VIG and Vig2 (Figure 7A). Similarly anti-VIG and anti-Vig2 antibodies pulled down both HP1 and HP2L (Figure 7B). Because VIG and Vig2 contain RNA binding domains, we tested whether these interactions with HP1 PLoS ONE | www.plosone.org 7 July 2009 | Volume 4 | Issue 7 | e6182