The reason for this could be the close proxi mity to euchromatin and one might expect such a beha vior also for W4. W3, W5 and W8 can be found in both the pericentromeric and the frontier region. enzalutamide mechanism of action W13 is loca lized roughly in the middle of the heterochromatic part of the q arm. Results are summarized in Table 2. Copy num ber estimates correspond to what was found in the literature 500 to 1,000 copies per genome for W1 and 20,000 to 200,000 for SMAlphafem 1. Several of the female specific repeats are transcribed in larvae but not in adults EST data suggested that some of the repeats could be transcribed and transcription of W1 and SMAlphafem 1 was described for cercaria. We extracted RNA from different life cycle stages and quantified the transcription level for repeats W3, W4, W5 and SMAlphafem 1.

For repeats W3 we did not find significant transcription above background. however, repeats W4, W5 and SMAlphafem 1 are transcribed in the larval stages. No transcripts could be detected in adult couples or immature females. At the genomic DNA level, we observed 20% differences in repeat copy numbers between different biological sam ples, but we did not observe a decrease in copy number, that is, shrinking of repeats, during the life cycle. Absence of transcription in adults is not, therefore, due to absence of repeats in the genome. The chromatin structure around the female specific repeats changes during the life cycle Repeat transcription has been linked to chromatin struc tural changes. We therefore analyzed histone isoforms that could potentially be associated with the female specific repeats.

Chromatin immunoprecipitation followed by mas sive sequencing was used to analyze the abundance of acetylated histone H3K9 around the repeats in miracidia, cercariae and adult couples. All 36 female specific repeats show a characteristic gradual decrease in H3K9 acetylation level from the larval stages to the adult stages. Among the total of 8,594 repeats in the genome, only 1,113 repeats show such a gradual decrease in H3K9 acetylation. The probability that such a pattern could be observed by chance for all 36 W specific repeats is negligible. To verify the ChIP Seq data by ChIP combined with qPCR, we focused on two transcribed repeats and one non transcribed repeat.

We used antibodies against H3K9Ac, tri methylated H3K4 that are characteristic for actively tran scribed euchromatin, and the heterochromatin markers tri methylated H3K9, and tri methylated H3K27. A region in the body of the alpha tubulin gene was used as reference for calculating the relative amount of immunoprecipitated DNA. The results are shown in Figure 4. Both euchromatic markers are enriched at the repeats in the miracidia stages where transcription was observed. In contrast, there are much fewer euchromatic markers around Cilengitide the repeats in adults. In the qPCR based experiments, cercariae occupy an intermediate position.