Drosophila RISC Component VIG and Its Homolog Vig2 Impact Heterochromatin Formation Elena Gracheva1¤a, Monica Dus2¤b, Sarah C. R. Elgin1* 1 Department of Biology, Washington University in St Louis, St Louis, Missouri, United States of America, 2 Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, United States of America Abstract Heterochromatin formation plays an important role in gene regulation and the maintenance of genome integrity. Here we present results from a study of the D. melanogaster gene vig, encoding an RNAi complex component and its homolog vig2 (CG11844) that support their involvement in heterochromatin formation and/or maintenance. Protein mutations vigEP812 and vig2PL470 act as modifiers of Position Effect Variegation (PEV). VIG and Vig2 are present in polytene chromosomes and partially overlap with HP1. Quantitative immunoblots show depletion of HP1 and HP2 (large isoform) in isolated nuclei from the vigEP812 mutant. The vig2PL470 mutant strain demonstrates a decreased level of H3K9me2. Pull-down experiments using antibodies specific to HP1 recovered both VIG and Vig2. The association between HP1 and both VIG and Vig2 proteins depends on an RNA component. The above data and the developmental profiles of the two genes suggest that Vig2 may be involved in heterochromatin targeting and establishment early in development, while VIG may have a role in stabilizing HP1/HP2 chromatin binding during later stages. Citation: Gracheva E, Dus M, Elgin SCR (2009) Drosophila RISC Component VIG and Its Homolog Vig2 Impact Heterochromatin Formation. PLoS ONE 4(7): e6182. doi:10.1371/journal.pone.0006182 Editor: Axel Imhof, University of Munich and Center of Integrated Protein Science, Germany Received February 10, 2009; Accepted May 29, 2009; Published July 8, 2009 Copyright: 2009 Gracheva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grant GM73190 to SCRE. M.D. was an Engelhorn Fellow of the Watson School of Biological Sciences. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: selgin@biology.wustl.edu ¤a Current address: Medros Inc, St Louis, Missouri, United States of America ¤b Current address: Department of Molecular Neurobiology, Skirball Institute, New York University, New York, New York, United States of America Introduction The nuclear content of a cell can be roughly divided into two categories: euchromatin and heterochromatin. Euchromatin, where most of the actively transcribed genes reside, is composed of a relaxed array of nucleosomes with corresponding epigenetic marks [1]. Heterochromatin, in contrast, is relatively condensed, as a result of interactions between biochemically modified histone tails and characteristic non-histone proteins, the components of a repressive chromatin assembly. The most prominent heterochro- matic mark is histone 3 di- and tri-methylation at lysine 9. This histone modification is found in a large variety of organisms and provides a platform for Heterochromatin Protein 1 (HP1) binding. HP1 recognition of the H3K9 methyl mark and interactions between the H3K9 methyltransferase Su(var)3-9 and HP1 are thought to enable spreading of heterochromatin over significant distances and to explain the existence of vast heterochromatic territories at centromeres. While the interaction of HP1 and H3K9me2/3 appears to be a consistent feature of constitutive heterochromatin, variations on this theme may be found to promote silencing in other contexts (reviewed in [2–4]). The level of chromatin condensation appears to be critical for appropriate regulation of gene expression. When a gene is moved from its normal location to a domain with a different chromatin density, the result is a mosaic gene inactivation, known as Position Effect Variegation (PEV). In Drosophila several genes with visible phenotypes, for which inactivation does not affect viability, serve as excellent PEV reporters. Among these, the white gene is probably the most heavily exploited. Several genetic screens using a variegating white gene as a reporter have identified mutations that enhance or suppress PEV. This has allowed discovery of key structural and regulatory components of heterochromatin, includ- ing Su(var)3-9. Additional analysis of PEV modifiers has led to an estimate that as many as 150 genes affect chromatin-related gene silencing [5]. However, so far only a small portion of these genes have been thoroughly investigated. An unanswered question is how heterochromatin formation is targeted and maintained through cell division. In S.pombe heterochromatin assembly at the centromere regions has been linked to transcription of the centromeric repeats. The process of silencing involves interactions between nascent transcripts pro- cessed by the RNAi system and the constitutive components of heterochromatin [6]. In this organism, an elegant ‘self-reinforcing loop’ model explains the specificity of heterochromatin formation based on the role of the RNAi machinery (reviewed in [7–9]). RNA interference, discovered less than a decade ago [10], has quickly became recognized as an important regulator of gene expression from plants to worms and humans. Besides the enzymatic core comprised by the protein Ago2, the RNA Induced Silencing Complex (RISC), purified from Drosophila S2 cells, contains dFMRP, VIG and Tudor-SN [11–13]. Mutations in ago2 and dfmr1 have been shown to affect PEV in flies, suggesting a possible role in heterochromatin formation [14,15]. Suppression of PEV by a gene mutation is an ‘output’ indicating that the silencing chromatin structure is not properly formed. Questions remain as to whether this misregulation occurs at initiation or maintenance, PLoS ONE | www.plosone.org 1 July 2009 | Volume 4 | Issue 7 | e6182