5436 Nucleic Acids Research, 2007, Vol. 35, No. 16 of telomeres is realized by germ-line speciﬁc transpositions of HeT-A, TAHRE and TART LINE elements (45–47). The aub and spn-E genes are implicated in the control of HeT-A and TART expression, accumulation of corre- sponding rasiRNAs and frequency of HeT-A and TART attachment to broken chromosome ends in ovaries (30). Thus, here we demonstrated the involvement of the rasiRNA pathway in the chromatin modiﬁcation of beneﬁcial telomeric retrotransposons and dangerous transposable elements. We found that elimination of rasiRNAs in Drosophila ovaries caused by spn-E1 leads to the decompaction of chromatin of retrotransposons. The decrease of HP1 level and the changes in histone modiﬁcation patterns, mani- festing itself in an increase of H3 K4me2 and decrease of H3 K9me3 were observed. A correlation between the most dramatic increase of HeT-A transcript abundance, owing to the spn-E1, piwi2 and armi1 mutations (Figure 3), and the most signiﬁcant changes of chromatin structure caused by spn-E1 compared to the I element, copia and copiaLTR- lacZ (Figure 2, Supplementary Figure 2) suggest that changes of chromatin structure in ovaries of rasiRNA mutants are accompanied by transcriptional activation of retrotransposons. At the same time, we detected no eﬀects of spn-E1 on the chromatin state of retrotransposons in somatic tissues. The observed germline speciﬁcity of rasiRNA-mediated retrotransposon silencing is in appar- ent contradiction with the observations that the spn-E, piwi and aub mutations aﬀect heterochromatin formation in somatic tissues (17,48) and these genes are required for variegated repression of a white reporter carried by the 1360 element (40). It is appropriate to point out that the size of the short RNAs corresponding to the 1360 element (40) and transgenic Fab-7 copies (39) ( 23 nt) in somatic tissues is consistent with Dicer-produced siRNAs, but not with rasiRNAs, suggesting that silencing of mobile elements in somatic tissues may be realized via RNAi, but not the rasiRNA pathway. Alternatively, rasiRNA- dependent heterochromatin formation might be induced in early stages of embryonic development and then be epigenetically inherited in somatic tissues in a rasiRNA- independent manner. The mechanism of chromatin modiﬁcation caused by rasiRNAs remains obscure. Although the Piwi protein was shown to be localized in cell nuclei (4,6,42), we failed to detect Piwi in the chromatin of retrotransposons (data not shown). Possibly, Piwi is associated with the nascent RNA but may easily leave chromatin. It has been suggested that rasiRNAs direct cleavage of retrotranspo- son transcripts (4–6). We propose that slicing of the nascent transcript mediated by the Piwi protein is capable to transform RNA polymerase II to a silencing complex. A similar model has been put forward to explain the spreading of transcriptional silencing in ﬁssion yeast Schizosaccharomyces pombe. It has been suggested that the sliced nascent transcript might recruit the silencing machinery to perform chromatin modiﬁcation (49,50). Further experiments are required to verify this model of chromatin silencing in the D. melanogaster genome. Our observations emphasize the proposed role of rasi(pi)RNAs in the formation of heterochromatin enriched by mobile elements and other repeats. Heterochromatin serves as a genome region to recruit and spread regulatory proteins to control chromosomal processes, including transcription as well as chromosome segregation. Actually, the disturbance of silencing of the Stellate repeats in the D. melanogaster genome is accompanied by chromosome meiotic non-disjunctions (51–53) and was shown to be triggered by spn-E mutations (22,51). Interestingly, spn-E mutations also lead to breakages in ovarian chromosomes (44) that might be caused by chromatin opening. The peculiarities of rasiRNA-dependent chromatin modiﬁcation in Drosophila male and female germinal cells require further detailed studies taking into account the known role of heterochromatin in chromosome mechanics (54). SUPPLEMENTARY DATA Supplementary Data are available at NAR Online. ACKNOWLEDGEMENTS We thank Alla Kalmykova for technical support in isolation of ovarian RNA used for short RNA cloning, Olesya Sokolova and Alina Korbut for help in RT-PCR and ChIP experiments, E.G. Pasyukova for kindly providing ﬂies carrying the copiaLTR-lacZ construct and James M. Mason for help in the manuscript preparation. This work was supported by RAS program for Molecular and Cell Biology, Russian Foundation for Basic Research (05-04-48034), the program of Scientiﬁc School support (6113.2006.4) and grant of President of Russian Federa- tion for young scientists (02.120.11.9326). Funding to pay the Open Access publication charges for this article was provided by Russian Foundation for Basic Research (05-04-48034). Conﬂict of interest statement. None declared. REFERENCES 1. Aravin,A.A., Lagos-Quintana,M., Yalcin,A., Zavolan,M., Marks,D., Snyder,B., Gaasterland,T., Meyer,J. and Tuschl,T. (2003) The Small RNA proﬁle during Drosophila melanogaster development. Dev. Cell, 5, 337–350. 2. Aravin,A.A., Klenov,M.S., Vagin,V.V., Bantignies,F., Cavalli,G. and Gvozdev,V.A. (2004) Dissection of a natural RNA silencing process in the Drosophila melanogaster germ line. Mol. Cell Biol., 24, 6742–6750. 3. Vagin,V.V., Sigova,A., Li,C., Seitz,H., Gvozdev,V. and Zamore,P.D. 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