Nucleic Acids Research, 2007, Vol. 35, No. 16 5433 Figure 2. ChIP analysis of retrotransposons in ovarian chromatin. TAF1 occupancy and histone modiﬁcations in the chromatin of spn-E1/spn-E1 (light bars) and spn-E1/+ ovaries (dark bars) were tested. DNA in precipitates was measured by quantitative real-time PCR using primers to promoter and coding regions (ORF) of LINEs (HeT-A and I element) and LTR retrotransposon copia. The level of H3 K4me2 histone modiﬁcation typical of transcriptionally active chromatin is signiﬁcantly lower in retrotransposons, than in ORF of the constitutive rp49 gene. The spn-E1 mutation increases the level of H3 K4me2 and decreases the level of H3 K9me3 and HP1 both in promoters and ORFs of retrotransposons, but not in chromatin of rp49 and Adh genes. The obtained data were normalized to the fragment of intergenic non-transcribed spacer, located in the 60D region. The ampliﬁed spacer fragment contains no binding sites for any known chromatin proteins and does not belong to any repeat that may be silenced. We detected a signiﬁcant amount of heterochromatic protein HP1 in the chromatin of I element, HeT-A and copia retrotransposons in ovaries. spn-E1 caused reduction of HP1 content in retrotransposons, especially for HeT-A (Figure 2). The 4-fold decrease of HP1 level was observed in promoters and 6-fold decrease in coding regions of HeT-A. At the same time, mutations in the HP1-encoding gene, which are available only in hetero- zygous state, lead to a drastic accumulation of HeT-A transcripts (36,37). This indicates that even a 2-fold decrease of HP1 level is suﬃcient for HeT-A derepression and the observed loss of HP1 occupancy of HeT-A chromatin, owing to spn-E1, causes transcriptional activation. The most pronounced changes of the histone marks and HP1 level in HeT-A chromatin correlates with the most dramatic increase of HeT-A transcript level, compared to copia and I element in spn-E1 ovaries (Figure 3). rasiRNA-mediated chromatin silencing is restricted to the germline It was demonstrated that SPN-E, PIWI and AUB proteins are required for heterochromatin formation in somatic tissues of D. melanogaster (17). Some rasiRNA-pathway components have also been shown to be required for nuclear organization of a chromatin insulator (38), functioning of Polycomb chromatin complexes (39) and