5430–5438 Nucleic Acids Research, 2007, Vol. 35, No. 16 doi:10.1093/nar/gkm576 Published online 15 August 2007 Repeat-associated siRNAs cause chromatin silencing of retrotransposons in the Drosophila melanogaster germline Mikhail S. Klenov1, Sergey A. Lavrov1, Anastasia D. Stolyarenko 1 , Sergey S. Ryazansky1, Alexei A. Aravin2, Thomas Tuschl2 and Vladimir A. Gvozdev1,* 1Department of Molecular Genetics of Cell, Institute of Molecular Genetics, Russian Academy of Sciences, Moscow 123182, Russia and 2Howard Hughes Medical Institute, Laboratory of RNA Molecular Biology, the Rockefeller University, 1230 York Avenue, Box 186, New York, New York 10021, USA Received May 19, 2007; Revised July 15, 2007; Accepted July 15, 2007 ABSTRACT Silencing of genomic repeats, including trans- posable elements, in Drosophila melanogaster is mediated by repeat-associated short interfering RNAs (rasiRNAs) interacting with proteins of the Piwi subfamily. rasiRNA-based silencing is thought to be mechanistically distinct from both the RNA interference and microRNA pathways. We show that the amount of rasiRNAs of a wide range of retroelements is drastically reduced in ovaries and testes of flies carrying a mutation in the spn-E gene. To address the mechanism of rasiRNA-dependent silencing of retrotransposons, we monitored their chromatin state in ovaries and somatic tissues. This revealed that the spn-E mutation causes chromatin opening of retroele- ments in ovaries, resulting in an increase in histone H3 K4 dimethylation and a decrease in histone H3 K9 di/trimethylation. The strongest chromatin changes have been detected for telomeric HeT-A elements that correlates with the most dramatic increase of their transcript level, compared to other mobile elements. The spn-E mutation also causes depletion of HP1 content in the chromatin of transposable elements, especially along HeT-A arrays. We also show that mutations in the genes controlling the rasiRNA pathway cause no dere- pression of the same retrotransposons in somatic tissues. Our results provide evidence that germinal Piwi-associated short RNAs induce chromatin modifications of their targets. INTRODUCTION A high level of transposable element expression is usually deleterious for the organism, leading to mutations and chromosomal rearrangements. Therefore, activity of mobile elements is thought to be under keen cellular control. Silencing of Drosophila selﬁsh elements is realized through the short RNA species, called repeat associated short interfering RNAs (rasiRNAs) (1–5) and also Piwi- interacting RNAs (piRNAs) (6). piRNAs play evolutio- narily conserved roles in the regulation of transposable elements in insects, mammals and zebraﬁsh (7–9) and are accumulated speciﬁcally in the germline (9–12). In Drosophila, the rasiRNA pathway requires members of the ‘Piwi subfamily’ of Argonaute proteins Piwi, Aubergine (Aub) and Ago-3 (2–6) but not the ‘Argonaute subfamily’ members, Ago1 or Ago2 (3), which guide microRNA and siRNA functions, respectively (13). rasiRNAs of 24–28 nt in length are longer than 21–22 nt siRNAs derived from dsRNA or 21–23 nt endogenous microRNAs (1,2). The increased length of rasiRNAs has aroused a suggestion of a peculiar mechan- ism of their formation (2). In ﬂies neither Dicer-1, which makes microRNAs, nor Dicer-2, which produces siRNAs, are implicated in rasiRNA formation (3). Recent publica- tions support a model in which discrete heterochromatic loci produce rasiRNAs that are predominantly antisense to transposons. The antisense rasiRNAs are bound by Piwi and Aub proteins and guide formation of sense rasiRNAs by cleavage of sense transposon transcripts (5,6). It was demonstrated that short interfering RNAs are implicated in chromatin modiﬁcations, such as methyl- ation of histone H3 K9, in yeast, plants and animal somatic cells (14–17). However, it remains unknown *To whom correspondence should be addressed. Tel: 09 51 96 00 12; Fax: 09 51 96 02 21; Email: gvozdev@img.ras.ru Present address: Alexei A. Aravin, Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, New York, 11724, USA. 2007 The Author(s) This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.