Characterization of occupancy across time of ERα and selected coregulators at ERα binding sites associated with E2-repressed genes. (A) UCSC Genome Browser schematics of selected ERα binding sites according to Carroll et al. (5). TSS, transcription site. (B to F) ChIP assays were performed at various times of 1 nM E2 treatment, using specific antibodies against ERα (B), p300 (C), CBP (D), SRC-3 (E), NRIP1 (F), or IgG negative control antibody. Data are averages ± standard errors of the means of the results of four or more independent experiments and are represented as the recruitment index (specific antibody signal/IgG signal ratio).

ERα transiently increases gene transcription at E2-repressed genes. (A) MCF-7 cells were treated with 10 nM E2, and nascent mRNA was labeled with biotin-UTP, isolated, and quantified via qPCR using 36B4 as the internal control. Data shown are means ± standard errors of the means of the results of three independent experiments. (B) RNA polymerase II ChIP was performed after 2 h of treatment with 5 μM α-amanitin followed by 1 h of 1 nM E2 treatment (α-ama+E2) or vehicle (α-ama). Change for α-ama+E2 versus α-ama is indicated above the red bars. Data are means ± standard errors of the means of the results of three independent experiments. Veh, vehicle.

p300 is required for CtBP1 recruitment, and CtBP1 is important for changes in histone marks. (A and B) ChIP assays were performed on MCF-7 cells after knockdown of p300 (A), CtBP1 (B), or GL3 control (A and B) with siRNA for 72 h. Antibodies for CtBP1 (A) or p300 (B) were used. (C) ChIP assays for histone marks (H3K14ac and H3K9ac) were performed after 45 min of 1 nM E2 or vehicle treatment, and data were normalized to total histone H3 content. (D) Histone H3K9ac changes were evaluated after CtBP1 or control GL3 (luciferase) knockdown as described for panel A. In panels A, B, and C, an asterisk indicates a P value of <0.05 for results of E2 treatment versus results for vehicle control. In panel D, an asterisk indicates a P value of <0.05 versus results for siGL3 vehicle. Veh, vehicle.

Characterization of early estradiol (E2)-repressed genes in MCF-7 breast cancer cells. (A) Time course of E2 regulation. MCF-7 cells were treated with 1 nM E2, and RNA was isolated at various time points. cDNA was measured by qPCR using primers for the genes indicated and internal control 36B4. (B) ICI182,780 antagonist experiments. MCF-7 cells were treated with 1 μM ICI182,780 (ICI) with or without 1 nM E2. (C) ERα siRNA experiments. MCF-7 cells were transfected with siRNA for ERα (siERα) or for GL3 control (siGL3) (luciferase) for 48 h prior to treatment with 1 nM E2. (D) Protein translation inhibitor experiments. MCF-7 cells were treated with cycloheximide (10 μg/ml) (CHX) with or without 1 nM E2. (E) Transcriptional inhibitor experiments. MCF-7 cells were treated with actinomycin D (5 μg/ml) (ActD) with or without 1 nM E2. In the experiments whose results are shown in panels B to E, the 1 nM E2 treatment was for 4 h, and total RNA was extracted and quantified by qPCR using 36B4 as the internal control. (F) RNA polymerase II ChIP was performed as described in Materials and Methods after 15, 25, or 45 min of 1 nM E2 treatment. Data are represented as the recruitment index (RNA polymerase II signal/IgG signal ratio). Data shown in all panels are averages ± standard errors of the means of the results of three or more independent experiments. In all panels, an asterisk indicates a P value of <0.05 for results of E2 treatment versus results for vehicle control. In panel D, a dagger indicates a P value of <0.05 for results for cycloheximide with E2 versus results for cycloheximide alone. Veh, vehicle.

p300 knockdown prevents E2-induced gene repression and E2-induced gene stimulation. (A) MCF-7 cells were transfected with siRNA against p300, CBP, or control GL3 luciferase for 72 h prior to 0.1% control ethanol vehicle or 1 nM E2 treatment for 4 h. p300 and CBP levels were assessed by immunoblotting. WB, Western blotting. (B and C) RNA was extracted after GL3, p300, or CBP siRNA treatment of cells followed by control vehicle or E2 treatment for 4 h, and the levels of E2 target genes were evaluated by qPCR. Data are means ± standard errors of the means of the results of three independent experiments and are expressed relative to results for GL3 siRNA vehicle-treated cells, set at 1. Veh, vehicle; *, P < 0.05 for results of E2 treatment versus results for vehicle control.

CtBP1, in a complex with p300, is required for E2-mediated gene repression. (A) ChIP assay using CtBP1 antibody was performed on MCF-7 cells after 45 min of control (0.1% ethanol) vehicle or 1 nM E2 treatment. (B) ChIP/reChIP was performed on MCF-7 cells after 45 min of 1 nM E2 treatment, using p300 antibody for the first pull-down and CtBP1 antibody or IgG control for the second pull-down. (C and D) CtBP1, CtBP2, or GL3 control siRNA was transfected into MCF-7 cells for 72 h prior to treatment with 1 nM E2 for 4 h. mRNA levels of target genes were measured by qPCR, and Western immunoblotting (WB) was used to confirm CtBP protein knockdown. Data are means ± standard errors of the means of the results of three independent experiments. In panels A, B, and D, an asterisk indicates a P value of <0.05 for results of E2 treatment versus results for vehicle control. For panel C, an asterisk indicates a P value of <0.05 versus results for GL3 siRNA. Veh, vehicle; si, siRNA.

Proposed model for ERα-mediated repression of early target genes. Following E2 treatment, ERα is initially recruited to ERα binding sites of both E2-stimulated and E2-repressed target genes, either via direct DNA binding or tethering to pioneer transcription factors (TF), where it transiently recruits coactivator complexes such as p300 and causes an increase in transcriptional output. After this early transient complex, ERα can maintain transcriptional activation by serving as a more-stable nucleation site for coactivator proteins, leading to histone acetylation (Ac) and engagement of RNA polymerase II (Pol II), or ERα can cause transcriptional repression by recruiting, via p300, CtBP1-containing repressor complexes which lead to RNA polymerase II dismissal and histone deacetylation. The change in color intensity for individual factors indicates the change in occupancy in the transcriptional complex over time.