Inhibition of eRNA production by flavopiridol does not inhibit ESR1, Pol II, or coregulator binding, alter H3K4me1 or H3K27ac levels, or prevent enhancer looping at ERBSs in MCF-7 cells. Locus-specific assays for E2-responsive enhancers showing the effects of a 1-h pre-treatment with flavopiridol (FP) on various molecular outcomes in MCF-7 cells. Each bar represents the mean + the SEM for three or more independent biological replicates. (A,B) Treatment with flavopiridol inhibits the E2-dependent production and steady-state accumulation of eRNAs and target gene mRNAs. RT-qPCR analyses for selected eRNAs and mRNAs in response to E2 treatment. (A) ERBS1 eRNA/P2RY2 mRNA and (B) ERBS2 eRNA/GREB1 mRNA. (C,D) ChIP-qPCR analyses for ESR1 (left) and Pol II (right) for ERBS1 (C) and ERBS2 (D) in the absence or presence of E2 and flavopiridol, as indicated. (E,F) ChIP-qPCR analyses for CREBBP (left), EP300 (middle), and Pol II (right) for ERBS1 (E) and ERBS2 (F) in the absence or presence of E2 and flavopiridol, as indicated. (G,H) ChIP-qPCR analyses for H3K4me1 (left), H3K27ac (middle), and H3 (right) for ERBS1 (G) and ERBS2 (H) in the absence or presence of E2 and flavopiridol, as indicated. (I,J) 3C-PCR analyses showing that looping between distal ERBSs and target genes in the presence of E2 is not blocked by flavopiridol (FP). (I) ERBS1/P2RY2 and (J) ERBS2/GREB1. The lowercase letters correspond to the primers denoted by orange arrows shown in Figure 1A. The assays were conducted in the presence (experimental) or absence (control) of DNA ligase, as indicated. The size of the PCR fragments in base pairs is shown. One representative experiment from three conducted is shown.
