Bidirectional Transcription Directs Both Transcriptional Gene Activation and Suppression in Human Cells (original) (raw)
Figure 3
Suppression of p21 sense/mRNA expression results in the recruitment of Ago-1 and H3K27me3 to the p21 promoter.
(A) siRNA p21-si858, which is targeted to the p21 sense/mRNA, suppresses p21 sense/mRNA transcription with no effect on p21 antisense transcription. Results from triplicate transfected MCF-7 cell cultures are shown with the standard deviations and p values from paired T tests. (B) The suppression of p21 sense transcription results in an increase, 24 hrs post-siRNA transfection, in Ago-1 at the p21 promoter whereas suppression of the p21 antisense transcript results in a loss of Ago-1 enrichment at the p21 promoter. Triplicate measurements from a single ChIP assay are shown with the standard deviations and p values from paired T tests. (C) The suppression of p21 sense transcription results in an enrichment of Ago-1 and silent state epigenetic marks H3K27me3, specifically at the p21 promoter 48 hrs post-siRNA treatment. The averages with the standard error of the means are shown from duplicate transfected cultures and p values from paired T tests. (D) Suppression of p21 sense/mRNA transcription results in an increase in transcription at the p21 promoter loci as determined by nuclear run on analysis. A dot blot and Image J analysis is shown from 2 separate experiments pooled together. The averages, standard deviations, and p values from paired T tests are also shown from the Image J analysis. (E) Suppression of p21 sense/mRNA transcription results in an increase in RNAPII p21 antisense transcriptional activity. Nuclear run-on analysis measured by quantitative RTPCR are shown with the averages from 2 separate experiments with duplicate treated cultures assessed spanning 24–48 hrs post-siRNA treatment. The standard error of means shown along with p values from paired T tests.