whether chromatin-based silencing of selﬁsh elements may be realized in the germline by Piwi-interacting RNAs, in particular by rasiRNAs in ﬂies. We show that the signiﬁcantly reduced abundance of rasiRNAs derived from a wide range of transposable elements in spn-E mutant ovaries is accompanied by the increase of H3 K4 dimethylation, decrease of H3 K9 di/trimethylation and depletion of HP1 content in the chromatin of retrotransposons. We demonstrate that rasiRNA-mediated silencing of tested retrotransposons takes place in ovaries, where it is necessary to protect the genome against transposon-induced mutations in progeny, but not in somatic tissues. MATERIALS AND METHODS Drosophila strains Strains bearing spn-E1, piwi2 and armi1 mutations were ru1 st1 spn-E1 e1 ca1/TM3, Sb1 es (point mutation in helicase domain of Spn-E), ru1 st1 spn-Ehls3987 e1 ca1/TM3, Sb1 es (P-element insertion into spn-E) (18,19), piwi2/CyO (P-ry11 transposon insertion) (20) and armi1/TM3 (P-element insertion) (21), respectively. P-element transformed ﬂies carrying the copiaLTR-lacZ construct were kindly pro- vided by E. G. Pasyukova. Discrimination in X-gal staining experiment of homo- and heterozygous larvae carrying spn-E1, piwi2 and armi1 mutations was done using GFP-expressing balancers CyO, P{w+m = hsp70: GAL4}P{w+m = UAS: GFP} and TM3, Sb1 es P{w+m = hsp70: GAL4}P{w+m = UAS: GFP}. RT–PCR analysis Total RNA was isolated from dissected ovaries or carcasses using Trizol reagent (Gibco BRL). The ﬁrst strand of cDNA was synthesized using SuperScript II reverse transcriptase (Gibco BRL) and oligo(dT) primer or speciﬁc primer according to the manufacturer’s instructions. cDNAs were analyzed by real-time quanti- tative PCR using SYBR Green. For PCR the following primers were used: 50-CCGTGGTCAACTTCACCAG CTC-30 (adh d2) and 50-TCCAACCAGGAGTTGA ACTTGTGC-30 (adh r2), corresponding to GenBank sequence AE003410.1 for Adh gene; 50-TCCGCCCAG CATACAGGC-30 (rp49 s2) and 50-CAATCCTCGTTGG CACTCACC-30 (rp49 as2), corresponding to GenBank sequence Y13939 for rp49 gene; 50-GCATGAGAGG TTTGGCCATATAAGC-30 (cop-s) and 50-GGCCCACA GACATCTGAGTGTACTACA-30 (cop-as), correspond- ing to GenBank sequence XO4456 for copia; 50-CGCAA AGACATCTGGAGGACTACC-30 (Het-s2) and 50-TGC CGACCTGCTTGGTATTG-30 (Het-as2), corresponding to GenBank sequence U06920 for HeT-A; 50-TGAAA TACGGCATACTGCCCCCA-30 (I el s2) and 50-GCTG ATAGGGAGTCGGAGCAGATA-30 (I el as2), corre- sponding to GenBank sequence M14954 for I element. X-gal staining and b-gal activity assay X-gal staining and b-gal activity assays were performed according to protocols described previously (2,22). Nucleic Acids Research, 2007, Vol. 35, No. 16 5431 Samples containing 5–15 pairs of ovaries dissected from 1 to 3-days-old females or 4–15 carcasses were used for b- gal activity assay. Measurements of b-gal activity were normalized to the total protein evaluated by the Bio-Rad protein assay kit. Short RNA cloning and annotation RNA preparation was performed as previously described (23). Total RNA was isolated from adult ovaries and testes. Cloning of miRNAs was performed as described (24). Characterization of cloned small RNAs was performed using local NCBI-BLAST 2.2.13 (25) against the canonical sequences of transposable elements (http://www.fruitﬂy.org/p_disrupt/datasets/ ASHBURNER/D_mel_transposon_sequence_set.fasta); Su(Ste) repeats (GenBank accession no. X59157|H-, Z11734|H- and Z11735|H-); miRNAs (http://microrna. sanger.ac.uk/sequences/, Release 8.0), tRNA (http:// lowelab.ucsc.edu/GtRNAdb/Dmela/) and rRNA (GenBank accession no. M21017). Only hits with 95% and higher similarity to transposable elements and Su(Ste) sequences and 100% similarity to other sequences were used. Parsing of results was done using corresponding BioPerl modules (26). Chromatin IP assay Ovaries were dissected from 1 to 10-days-old females in 1X PBS and stored in 1.5 ml tube on ice during isolation (up to 2 h). PBS solution was removed after centrifugation (3500 r.p.m. 1–2 min). 10 mg of material (about 150 ovaries or 100 carcasses) was used for one IP reaction. The chromatin IP assay was performed as described previously (27), using polyclonal rabbit antibodies (Upstate): Anti-dimethyl-Histone H3 Lys4 (#07-030), Anti-dimethyl-Histone H3 Lys9 (#07-441), Anti- trimethyl-Histone H3 Lys9 (#07-523) and anti-HP1 (PRB-291C Covance innovative). Anti-TAF1 was kindly provided by G. Cavalli. DNA precipitates were ampliﬁed by semiquantitative PCR in the presence of aP32 dATP or real-time quantitative PCR. PCR product quantities were normalized to input and relations to a fragment of intergenic spacer in the 60D region were calculated. No identiﬁed or predicted genes are located 2.5 kb upstream and 4.3 kb downstream of the 60D ampliﬁed fragment. The TRANSFAC database search found no binding sites for any known chromatin proteins and transcriptional factors in the fragment. Final enrichment values of sample PCR products were calculated using the following expression: E(product)sample E(60D)input/E(60D)sample E(product)input. The following primers were used for PCR analysis in ChIP: 50-CAACA CTACTTTATATTTGATATGAATGGCC-30 and 50- CGAAAGGGGGATGTGCTGC-30 for ampliﬁcation of the promoter region of copiaLTR-lacZ construct; 50- CAACACTACTTTATATTTGATATGAATGGCC-30/ 50-GCGTACTTCTCGCCATCAAACG-30 and cop-s/ cop-as (see above) for endogenous copia promoter region and ORF, respectively; 50- ACCACGCCCAACCCCCAA-30/50-GCTGGTGGAGG TACGGAGACAG-30 and Het-s2/Het-as2 (see above),