Fig 1: E2-Repressed eRNAs Facilitate the Formation of a Functional ERa-Centered Transcriptional Complex at E2-Downregulated Enhancers(A) Expression of indicated RNA molecules after KDM2A knockdown in MCF-7 cells treated with 100 nM E2 for 3 h.(B) ChIP of Pol II and KDM2A at specified enhancers at different time points upon 100-nM E2 treatment in MCF-7 cells. ChIP with normal IgG antibody was included as a negative control.(C and D) ChIP of KDM2A (C) or Pol II (D) upon knockdown of TM4SF1 eRNA1. MCF-7 cells were treated with (+) or without (-) 100 nM E2 for 5 min (C) or 30 min (D).(E) In vitro association between KDM2A protein and ERa-Fr-2 protein in the presence of increasing amount of TM4SF1 eRNA1.(F) In vitro competitive binding assays by mixing the in-vitro-transcribed eRNAs and the double-stranded PCR products containing enhancer regions in the presence of purified recombinant proteins. The precipitates from cobalt resin were subject to either western blot or ethidium bromide (EtBr) staining.Data in (A)–(D) are presented as mean ± SD with three biological and technical replicates. Statistical significance in (A), (C), and (D) was calculated using t test. *p < 0.05; **p < 0.001. Also see Figure S8.