Fig2. Importance of eRNA for target gene activation.

(a,b) siRNA/LNA knock-down of eRNAs. Efficacy and effects on coding gene transcription assessed by QPCR for the TFF1, FOXC1, and CA12 eRNAs and coding transcription units. (c) QPCR analysis showing no significant change of several E₂-target coding genes when FOXC1 eRNA was knocked-down using LNA. (d) Lack of effect of NRIP1, TFF1, or CA12 eRNA knock-down on expression of other coding genes located much more distal, including USP25 (520kb from NRIPe), RSPH1, (120kb from TFF1e) and APHIb (110kb from the CA12e). (e) GRO-seq data from FOXC1e LNA treated cells showing its inhibitory effect on the transcription of FOXC1 coding locus, but not on the targeted enhancer region itself. The bar graph (right) shows that the FOXC1e LNA knocked-down E₂-induction of FOXC1 mRNA (tag counts over the whole gene length), but not transcription of the enhancer region. (f) A similar snapshot as in panel e showing the lack of effect from FOXC1e LNA on GAPDH transcription. Data represent mean±SEM (a,b) and mean±SD (c,d). (n=3). *p<0.05, **p<0.01.