Fig4. Role of eRNA in Cohesin-dependent gene activation.

(a) Co-immunoprecipitation of Rad21 and SMC3 with ERα from E₂ or ethanol treated MCF7 whole cell extracts showing physical interaction between ERα and Cohesin subunits, which is further enhanced by E₂ treatment. Numbers below blots depict the band density (Image J) relative to that of corresponding density of ERα. (b) Rad21 enrichment centered at UP-enhancers as determined by ChIP-seq, which shows moderate E₂-induced increase. (c) In vitro transcribed (IVT) RNA pull-down assay showing the interaction between Cohesin subunits and eRNAs, but not a control RNA (RNA fragment of Xenopus elongation factor α). (d) RIP-QPCR showing binding of Rad21 to selected regulated eRNAs but not to GAPDH or TUG1. (e) ChIP-QPCR analyses represent the inhibitory effect from knock-down of NRIP1e (siRNA and LNA), FOXC1e or TFF1e on E₂-indcued Rad21 additional recruitment, but not on H3K4me¹. (f) Effect of Rad21 depletion on physical interaction between promoter:enhancer for the GREB1 and NRIP1 genes, assessed by 3C assay. (g) 1,309 E₂-induced coding genes were defined by GRO-seq from siCTL (±E₂) group and then their fold change (log2 FC) in siCTL versus siSMC3 transfected MCF7 cells was plotted). Mean ± SD. (n=3). *p<0.05, **p<0.01.