Epigenetic modifications post dsRNA transfection.

<p>(A)Chromatin remodeling after S1, S5 or S9 dsRNA transfection. Significant reduction in MNase digestion 72 hours after S1 and S5 transfection while increase in the accessibilty after S9 transfection in HeLa cells. Cells were transfected with the respective dsRNAs followed by MNase-PCR 72 hours post transfection. S1:S1 dsRNA transfected cells, S5-dsRNA: S5 dsRNA transfected cells, S9-dsRNA: S9 dsRNA transfected cells, Control-dsRNA: Control dsRNA transfected cells (*) P < 0.01. Bi-Biv. Enrichment of H3K9me2, H3K27me3 and H3K4me2 at dsRNA target sites. dsRNA induced TGS is associated with increase in H3K9me2 and H3K27me3 while TGA is associated with gain in H3K4me2. HeLa cells were transfected with respective dsRNAs followed by ChIP analysis 72 hours later using the desired antibodies to pull the target DNA. Precipitated DNA was analyzed by PCR using region specific primers. DNA pulled down in the absence of antibody (mouse IgG) served to identify background amplification while input DNA was amplified as a loading Control. (Bi) Gel electrophoretogram showing PCR products obtained after ChIP in S1 and S5 dsRNA transfected cells. (Biii) Gel electrophoretogram showing PCR products obtained after ChIP in S9 dsRNA transfected cells. (Bii and Biv) Densitometric analysis of ChIP data shown in A and B, obtained with ImageJ software. Relative enrichment = ChIP/Input DNA. 1: H3K9me2 immunoprecipitated PCR, 2 and I: H3K27me3 immunoprecipitated PCR, 3 and III: mouse IgG immunoprecipitated PCR, 4 and IV: Input DNA, II: H3K4me2 immunoprecipitated PCR. Ci-Cii. Effect of HDAC inhibition on TGS and TGA. Expression ratio of pRNA, E6 and E7 after S1, S5 or S9 transfection. HeLa cells were transfected with dsRNA (100nM) and 24 hours later treated with DMSO or TSA (400nM) followed by RNA isolation 48 hours after the treatment. (Ci) TSA treatment in dsRNA transfected cells. (Cii) DMSO (solvent for TSA and AZA) treatment in dsRNA transfected cells.