Figure 5

CCCTC-binding factor (CTCF) binding and H3K27 trimethylation (H3K27me3) at p21 (cyclin-dependent kinase inhibitor 1A gene, also known as CDKN1A ) in response to the CTCF proximal (cp)-transcription initiation (ti)RNA sponge and mimics. (a) Transfection with the cp-tiRNA sponge resulted in an enrichment of CTCF at the conserved CTCF binding site. H3K27me3 was unaffected at the canonical p21 promoter or in the p21-coding region. (b) Transfection with cp-tiRNA mimics resulted in a decrease of CTCF at the CTCF binding site. H3K27me3 was generally unaffected. (c) Transfection with the cp-tiRNA sponge resulted in a decrease of H3K27me3 at the alternate p21 promoter, consistent with an increase in transcriptional activity. (d) As measured by western blot, samples transfected with the cp-tiRNA sponge showed an increase of p21 protein levels, while samples transfected with the cp-tiRNA mimics showed a decrease. (e) Differences in p21 protein levels from the western blot in (d) quantified using ImageJ. (a-d) Samples were analyzed as indicated 72 h post transfection. No antibody values were subtracted from each immunoprecipitation, and the resultant values were standardized to the input each sample. The averages of samples transfected in triplicate are shown, error bars represent the standard errors of the means, and P values from paired t tests are shown.