Figure 7. Co-immunoprecipitation experiments show the association of heterochromatin structural proteins HP1 and HP2 with the RNA-interacting proteins VIG and Vig2. (A) The HP1 monoclonal antibody C1A9 pulls down HP2 large isoform (as expected) and both VIG and Vig2 proteins. Mouse IgG was used for the control mock immunoprecipitation. (B) Antibodies specific for VIG (CSH1801) and Vig2 (CSH2542) proteins pull down HP2 Large isoform and HP1. Rabbit IgG was used as a negative control. (C) RNaseA treatment of the homogenates reduces the amount of VIG and Vig2 VIG and Vig2 Impact Chromatin protein pulled down by the HP1 antibody. Mouse IgG was used for the control mock immunoprecipitation. doi:10.1371/journal.pone.0006182.g007 were dependent on an RNA component. Indeed, treatment with RNase A prior to immunoprecipitation with anti-HP1 antibodies resulted in loss of signal, suggesting that the HP1/ VIG and HP1/ Vig2 interactions are RNA dependent. Discussion We have presented new results suggesting the involvement of the RNAi-associated protein VIG, and its homolog Vig2, in heterochromatin formation. VIG and Vig2 belong to the PAI- RBP1 family. Based on DNA sequence and protein database analyses, one can infer that these proteins play a role in mRNA stability and/or in processes requiring interactions with RNA [26]. Human paralogs of VIG, SERBP1 and HABP4, have been reported to localize in the cell cytoplasm, nucleus, and perinuclear regions; our data indicate that VIG and Vig2 are both nuclear and cytoplasmic (Figure 2). Human VIG paralogs interact with the silencing chromatin remodeling complex CHD3, as shown by a yeast two-hybrid screen [44]. Our data expand the proposed role of PAI domain proteins in chromatin regulation by demonstrating that deficits in VIG and Vig2 can result in suppression of PEV (Figure 3). We observe both in vivo co-localization (Figure 4) and evidence of interaction (Figure 7) with major components of repressive chromatin, HP1 and HP2. It has recently been reported that in Drosophila, HP1 binding and H3K9 methylation in heterochromatin are dependent on physical interaction with ‘inactive’ (unphosphorylated) STAT [45]. This is a surprising connection, since canonical JAK/STAT regulation pathways have been thought of as generally involved in transcription regulation. VIGs’ human paralog SERBP1 interacts with Protein Inhibitor of Activated STAT (PIAS) [46], which has its own fly paralog encoded by the Su(var)2-10 gene [47]. These pieces of information suggest an interpretation of our results. Our data demonstrate that VIG is involved in stabilization of HP1 binding to chromatin (Figure 6) and that Vig2 affects H3K9me2 levels (Figure 5). Considering the findings discussed above, one can speculate that in Drosophila VIG and Vig2 might function in concert with components of the JAK/STAT pathway or a non- canonical STAT pathway to modulate heterochromatin forma- tion. In particular, RNA binding proteins such as VIG, perhaps collaborating with Su(var)2-10, a PIAS protein, could potentially facilitate binding of inactive STAT and consequently HP1 to heterochromatic domains. The Drosophila JAK/STAT pathway has an important role in the immune response, particularly in viral defense [48], a process also related to RNAi. Our results indicate that the association between VIG or Vig2 and HP1 is RNA dependent. One can speculate that transcripts originating from transposable elements (a major sequence component of hetero- chromatin), processed by the RNAi machinery, could be the constituents of a targeting complex containing HP1. VIG has been demonstrated to interact with small RNAs in Drosophila S2 cells. The human paralog of VIG has been shown to interact with L1 retrotransposon products in the stress granules and to participate in regulation of retrotransposition together with other RNAi components, including Ago2 and FMRP [49]. However, Drosophila VIG and Vig2 -interacting RNAs have yet to be identified and characterized. Many unanswered questions still remain. Why do deficits of either VIG or Vig2 have such different impacts on HP1 nuclear binding and H3K9 methylation? Many previous studies have PLoS ONE | www.plosone.org 8 July 2009 | Volume 4 | Issue 7 | e6182