whether the gene product is physically involved in heterochromatin formation, or whether the PEV outcome is a result of indirect effects. The RNAi effector protein Ago2 is associated with endogenous siRNAs targeting some transposons and protein coding genes in germ line and somatic cells [16–18]. Another member of Argonaute family, Piwi, is involved in piRNA generation in gonads [19]; piwi mutations suppress PEV and Piwi protein can interact with HP1 directly [20,21]. The large majority of piRNAs (Piwi complexes) and to a much less extent some endo siRNAs (Ago2 complexes) are derived from transposable elements and regulate their silencing in different compartments [16,17,22–24]. In Drosophila up to 77% of the recently annotated 24 Mb of heterochromatic sequences are classified as repetitive transposable elements (TEs). Among these, the retrotransposons (LTRs and LINEs) are the largest group [25] While much of the silencing due to RNAi is accomplished by a post- transcriptional mechanism, it is tempting to speculate that an RNAi- based mechanism may well be an essential part of targeting and maintaining heterochromatic structure in flies. Among the Ago2- RISC accessory components, VIG belongs to a family of proteins with a recognizable RNA binding motif (the PAI-RBP1 family) that was first identified in a HTC rat hepatoma cell culture. Computational analyses have revealed related proteins in other species including Homo sapiens, Drosophila melanogaster, and Arabodopsis taliana, but not in single cell organisms [26]. The D. melanogaster genome contains three genes that encode proteins with the PAI-RBP1 motif: VIG, CG11844 (which we will refer to as Vig2) and CG15031 [26]. Vig (vasa intronic gene) is located in an intron of vasa on the second chromosome and copurifies with Ago2 in a complex containing small RNAs [12]. Mutations disrupting vig cause up-regulation of expression from some retrotransposons in D.melanogaster ovaries [27], suggesting the possibility of involvement in endogenous siRNA regulation as a member of an Ago2 complex. Defects in the antiviral responses of vig mutant flies have also been reported [28]. In contrast very little is known about vig2 (third chromosome) and CG15031 (X chromosome), although CG15031 has been reported to act as a protein phosphatase Y-interacting protein [29]. Here we examine the role of VIG and its close homolog Vig2 in heterochromatin formation. We find that while mutations in both genes result in suppression of PEV, it appears that the two proteins impact heterochromatin structure through different mechanisms. Results Developmental expression profile of vig and vig2 VIG and Vig2 proteins are very similar in amino acid sequence (Figure S1), suggesting possible overlapping roles. To study vig and vig2 we generated developmental profiles of RNA and protein levels (Figure 1). Specifically, we were interested in studying material from the developmental stages when heterochromatin formation occurs. Some of the genes involved in RNAi and implicated in heterochromatin formation (piwi, homeless) are preferentially expressed in the germline and the proteins are maternally loaded. Heterochromatin formation begins at the blastoderm stage at approximately 1.5 hours of embryogenesis [30]. A model proposed by Eissenberg [31] suggests that it is stably maintained during mitotic proliferation from mid-embryogenesis to 3rd instar larvae, and than becomes more relaxed in the post- mitotic period (pupal and adult stages). We examined vig and vig2 expression in early embryos (2–4 h), late embryos (6–18 h), larvae (2nd and 3rd instars), and adults (somatic tissue and germline) using both real-time RT PCR and Western blots. To detect VIG and Vig2 proteins we generated polyclonal peptide antibodies. Anti-VIG (CSH1801) identifies a VIG and Vig2 Impact Chromatin Figure 1. A developmental profile of expression of the vig and vig2 genes generated using quantitative real-time RT PCR and Western immunoblotting. (A) Vig and vig2 are transcriptionally active throughout development reaching the highest levels in ovaries (vig2) and in larvae (vig). Vig and CG11844 expression levels shown are normalized to the expression of the RpL32 gene. (B) VIG protein can be detected at all stages of development and in somatic as well as in germline tissue (Western blot using CSH1801). Significant protein accumulation occurs in late embryogenesis and at the larval stage. Vig2 protein is at its highest levels in ovarian tissue and early embryos, but is present throughout development; the amount of protein gradually declines and becomes undetectable in adult soma (Western blot using CSH2542). H2B antibodies were used for the loading control. doi:10.1371/journal.pone.0006182.g001 specific band of ca 60 kDa on a Western, consistent with the previously reported molecular weight [12]. Anti-Vig2 (CSH2542) recognizes a protein of ca 50 kD. The specificity of the antibodies was verified in vivo by gene knock down in Drosophila cell culture (Figure S2). We observed differences in the expression patterns of vig and vig2 genes (Figure 1). Vig2 is activated earlier in development, with transcription peaking in ovaries, and accumu- lation of the protein in the germline and early embryo. Later the gene is transcribed at lower levels and the amount of protein declines. VIG is also present in the ovaries and early embryos, but transcription peaks at the late embryonic stage; protein accumu- lation starts in the late embryo and reaches its maximum amount in larvae, when the amount of Vig2 is reduced. While VIG protein is still present in adult somatic tissue, Vig2 is not detected. Cellular localization of VIG and Vig2 For better functional characterization we performed a partial purification of VIG and Vig2 proteins from nuclear and PLoS ONE | www.plosone.org 2 July 2009 | Volume 4 | Issue 7 | e6182