Decreased dCAP-D3 levels result in spreading of repressive histone marks and an opening of the chromatin at a dCAP-D3 regulated gene cluster containing a retrotransposon.

<p>A) ChIP for trimethylated H3K9 (a' and b') and trimethylated H3K4 (c' and d') performed on the <i>mdg1-1403</i> locus in SG4 cells treated with control dsRNA (black bars) demonstrates absent or low levels of the marks in the areas surrounding the retrotransposon but high levels of H3K9me3 within retrotransposon sequence. ChIP for trimethylated H3K9 in cells treated with dCAP-D3 dsRNA demonstrates a dCAP-D3 dependent increase of the mark in the areas surrounding the retrotransposon (a') and a dCAP-D3 dependent decrease of the mark in within retrotransposon sequence following retrotransposon mobilization (b'). ChIP for trimethylated H3K4 in cells treated with dCAP-D3 dsRNAs shows a dCAP-D3 dependent increase over the entire locus prior to mobilization (c') which persist following mobilization. Primer sets used are depicted above the charts. Primer sets “LTR” and “5” are not specific for each of the loci but instead prime global <i>mdg1</i> sequence. Results are the averages of 2 experiments involving duplicate IPs and are presented as a percentage of the IP with control IgG ChIP signal subtracted. (*) indicates a quantitative comparison between indicated ChIP signal in control dsRNA and dCAP-D3 dsRNA treated cells with a p-value less than 0.05 as calculated by student unpaired t-test. (+) indicates a quantitative comparison of specific ChIP signal to the average over the entire locus with a p-value less than 0.05 as calculated by student unpaired t-test. (B) qRT-PCR for transcript levels of genes surrounding <i>mdg1-1403</i> (as depicted in diagram in A) indicates that compared to control dsRNA treated cells, transcription is decreased in dCAP-D3 dsRNA treated cells prior to retrotransposon mobilization (day 4) and increases to almost normal levels on the day of retrotransposon mobilization (day 5). Following retrotransposon mobilization (day 6), transcription increases more than 2-fold in dCAP-D3 dsRNA treated cells. CG31343 is positioned approximately 12 kb upstream of <i>mdg1-1403</i>. Results are the average of three experiments and (*) indicates p-value less than 0.05 as calculated by student unpaired t-test.