5432 Nucleic Acids Research, 2007, Vol. 35, No. 16 Figure 1. The spn-E1 mutation leads to the decrease of the overall rasiRNA abundance in ovaries. Length distribution of cloned rasiRNA and the ratios of rasiRNA to microRNA amounts are indicated by bars. corresponding to HeT-A promoter region and ORF, respectively; 50-CGTGCCTCTCAGTCTAAAGCCTC- 30/50-CCCGGATTAGCGGTATTGTTGTT-30 and I el s2/I el as2 (see above), corresponding to I element promoter and ORF, respectively; adh d2 and adh r2 (see above), corresponding to Adh gene; rp49 s2 and rp49 as2 (see above), corresponding to rp49 gene; 50-CGGC GAGGGGGGAAAAGGAC-30 and 50-CTTGGCAGC AGGTGGAAAATGTT-30, corresponding to the 60D intergenic spacer. RESULTS The presence of rasiRNAs corresponding to a wide range of transposable elements requires Spn-E function The spn-E (spindle-E, homeless) gene encodes a putative DExH box RNA helicase, which is required for rasiRNA- mediated silencing of selﬁsh elements (28–30). Previously it was shown that the spn-E1 mutation leads to the loss of testis short RNAs related to the Su(Ste) repeats (2) and ovarian short RNAs of the HeT-A LINE element (30) and roo LTR retrotransposon (3). To address the eﬀect of the spn-E gene on total rasiRNA abundance, we cloned short RNAs from spn-E1 homo and heterozygous ovaries and testes (Supplementary Table 1). In ovaries the quantity of rasiRNAs was 5-fold higher than that of miRNAs. This is a drastically increased ratio compared to the one calculated previously for Drosophila embryos and adult ﬂies (about 0.65 and 0.1, respectively) (1). In contrast to ovaries, approximately equal amounts of microRNAs and rasiRNAs were observed in testes. The amount of rasiRNAs cloned from homozygous spn-E1 ovaries was 6.7 and 3.3 times lower than in heterozygotes if normal- ized to microRNA or to the sum of cloned fragments of ribosomal and transfer RNA, respectively (Figure 1). Both sense and antisense rasiRNAs abundance was decreased in spn-E1 homozygous ovaries. spn-E1 exerted the most pronounced eﬀects on the amount of rasiRNAs related to LINE elements (Doc, F-element, G2 and R1A1) and some LTR retrotransposons (GATE, gypsy6 and MAX-element). The total amount of LINE-related rasiRNAs normalized to miRNAs was 20-fold lower in homozygous spn-E1 ovaries, whereas only a 4-fold decrease of LTR retrotransposon rasiRNA abundance was revealed (Supplementary Table 1). Derepression of transposable elements in the germline correlates with opening of chromatin structure To investigate the role of chromatin state in rasiRNA- mediated transposable element silencing, we performed ChIP analysis of chromatin in ovarian nuclei using antibodies speciﬁc to known histone modiﬁcations. We focused on the three extensively investigated retrotran- sposons of Drosophila melanogaster: I element, HeT-A (LINE elements) and LTR-containing copia element. These three retrotransposons were shown to be up- regulated due to spn-E1 and other mutations, aﬀecting the rasiRNA pathway in ﬂies (3,28,30). In spn-E1/+ heterozygous ovaries the chromatin of promoter and coding regions of tested retrotransposons compared with that of the ORF of the ribosomal rp49 gene contained a signiﬁcantly lower level of histone H3 dimethylated at lysine 4 (H3 K4me2), the principal mark of transcriptionally active chromatin (31) (Figure 2). On the contrary, chromatin of retrotransposons was enriched with H3 K9me2 and particularly with H3 K9me3 mark, which are speciﬁc for inactive chromatin (32,33) (Figure 2). In spn-E1 homozygous ovaries we observed an increase in H3 K4me2 and a decrease in H3 K9me3 in promoters, as well as in coding regions of retrotranspo- sons, but not in the chromatin of rp49 and Adh genes (Figure 2). Since methylation of H3 K4 was shown to be a cotranscriptional process (34), the increase in H3 K4me2 in the chromatin of retrotransposon coding regions may be considered as a consequence of an elevated level of their transcription. Along with endogenous retroelements, we performed ChIP analysis of a transgenic construct containing the reporter lacZ gene driven by copia LTR (copiaLTR-lacZ) located on the X chromosome. We also observed an increase of H3 K4me2 occupancy in spn-E1 homozygous ovaries, but no decrease of the repressive H3 K9me2 and H3 K9me3 marks (Supplementary Figure 2). The absence of this latter eﬀect may be attributed to the euchromatic location of the copiaLTR-lacZ transgene compared to the mainly heterochromatic locations of endogenous copia elements. The level of TAF1 protein, which is a known component of RNA polymerase II transcription initiation complex TFIID (35), remained unchanged in spn-E1 homozygous ovaries in HeT-A and copia promoters. The TAF1 level was increased 3-fold in the I element promoter (Figure 2) and increased 2-fold in copiaLTR-lacZ transgenic construct (Supplementary Figure 2). These results allow us to propose that chromatin opening is unlikely to occur as a result of enrichment with basal transcription factors in promoter regions.