ERα activity depends on interaction and target site corecruitment with phosphorylated CREB1

(A) Transfection controls of the different exogenous forms of CREB1 expressed in MDA-MB-134 cells. The wild-type CREB1 and the phosphoserine mutant forms of CREB1 (CREB1-S133A and CREB1-S133D) have an HA tag, whereas the dominant-negative form (A-CREB1) has a FLAG tag. Representative immunoblots with antibodies against the indicated tags are shown. (B, C) Efficiency of CREB1 and ERα knockdowns performed in MDA-MB-134 cells. Representative RT-PCR analysis of reduction of CREB1 mRNA levels (B) and immunoblots of CREB1, pCREB1, and ERα (C).

(A, B) Luciferase reporter assays in HEK293T cells with exogenously expressed ERα. Cells were cotransfected with a luciferase reporter fused to either an ERE (A) or a CRE (B) and with CREB1 expression vectors as indicated. Cells were stimulated with vehicle alone (DMSO, indicated with a C), E2, or FI as indicated in the Materials and Methods section. All values marked with an asterisk are statistically significantly different from their respective vector controls with P-values < 0.05.

(A–C) CREB1 overexpression promotes the expression of the indicated ERα target genes. MDA-MB-134 cells were transfected with the indicated expression vectors and stimulated with vehicle, E2, or FI for 6 h before extraction of RNA for quantitative RT-PCR analysis of the indicated ERα target genes. See the Materials and Methods section for more experimental details. All values marked with an asterisk are statistically significantly different from their respective vector or siRNA controls with P-values ≤ 0.05; those marked with a small circle are significantly different from the S133D sample. (D) Effect of transient siRNA-mediated knockdown of CREB1 on the expression of a panel of ERα target genes, as determined by quantitative RT-PCR analysis; cells and treatments as in panels A–C. All values marked with an asterisk are statistically significantly different from their respective siRNA controls with P-values < 0.05.

(A) Schematic representation of ERα domains. (B–E) The ERα HBD functionally collaborates with CREB1. Luciferase reporter assays in HEK293T cells with fusion proteins of the DNA-binding domain of Gal4 with the indicated ERα domains: in panels B–E, the A/B domain containing the activation function AF1, the DNA-binding domain (domain C), the hinge (domain D), and the HBD with activation function AF2 (domains E/F). These were transiently coexpressed with wild-type CREB1, CREB1-S133A, and the dominant-negative CREB1 mutant (A-CREB1). (F) Luciferase reporter assay for Gal4-CREB1 activity in HEK293T cells. All values marked with an asterisk are statistically significantly different from their respective vector controls with P-values < 0.05.

(A–C) ChIP-qPCR assays to determine the importance of CREB1 and its phosphorylation on S133 for the recruitment of ERα to target genes. MDA-MB-134 cells were transfected and treated as indicated in the figure and in the Materials and Methods section. Note that the fold recruitment values for ERα are experimentally variable between different experiments; these values should only be compared between different treatments and conditions within the same experiment/panel. (D) ChIP-qPCR assays to determine the impact of a siRNA-mediated knockdown of CREB1 on the recruitment of ERα to a wider panel of target genes. CREB1 was knocked down in MDA-MB-134 cells before stimulation as indicated. All values marked with an asterisk are statistically significantly different from their respective vector or siRNA controls with P-values < 0.05.

(A–C) ChIP-qPCR assays to determine the importance of CREB1 and its phosphorylation on S133 for the recruitment of ERα to target genes. MCF7 cells were transfected and treated as indicated. (D) ChIP-qPCR assays to determine the impact of a shRNA-mediated knockdown of CREB1 (with a mixture of shRNA constructs three and four) on the recruitment of ERα. shRNA constructs were introduced as lentiviruses. (E) Efficiency of CREB1 knockdown performed in MCF7 cells with two separate mixtures of shRNA constructs. Immunoblots of CREB1 and pCREB1 with tubulin as loading control.

(A–C) Co-immunoprecipitation of endogenous ERα, CARM1, and pCREB1 as a function of stimulus. MDA-MB134 cells were stimulated with DMSO (indicated by a C), E2, or FI before cell lysis and immunoprecipitation as indicated in the figure and in the Materials and Methods section. Immunoprecipitations were performed with the antibodies indicated above the immunoblots, that is, either antibodies against ERα or CARM1 or a corresponding control antibody (IgG). Proteins revealed by immunoblotting are indicated on the left. (D–F) ERα as a function of stimulus. Exogenous HA-tagged wild-type and mutant CREB1 were immunoprecipitated from extracts of transfected MDA-MB-134 cells to assess the association with endogenous ERα by immunoblotting with an antibody against the HA tag or ERα.

(A, B) Average signals for the ChIP-seq datasets for ERα (A) and pCREB1 (B), obtained with MDA-MB-231 cells treated as indicated. The peak intensities are centered at the transcription factor peaks with a 2-kb window. (C, D) Venn diagrams of ERα (C) and pCREB1 (D) chromatin-binding sites as a function of treatment. Below the Venn diagrams, the number of sites (locations) that do not overlap between treatments are indicated. (E, F) Heat maps showing the ChIP-seq signals of ERα (E) and pCREB1 (F) in response to the indicated stimuli. Chromatin binding sites are sorted according to decreasing ChIP signals and centered at the respective transcription factor peaks with a 500-bp window on either side.

(A) Heat maps showing the ChIP-seq signals of pCREB1 over the ERα binding sites in response to the indicated stimuli. Regions are sorted according to decreasing signals in pCREB1 binding. Data are centered at the ERα peaks with a 500-bp window on either side. All data are from MDA-MB-134 cells. (B, C) Venn diagrams of the cAMP-induced sites (B) and the E2-induced sites (C) indicating those shared between ERα (red) and pCREB1 (blue). (D) Genomic distribution of the peaks shared between ERα and pCREB1 in response to the indicated treatments. (E) Snapshots from the ChIP-seq data for both ERα and pCREB1 peaks are shown across indicated genes for DMSO (blue), E2 (red), and FI (green). Genomic coordinates are indicated. The numbers in square brackets in each panel indicate the scale based on the numbers of reads.

(A, B) Genomic distribution of the unique peaks of either ERα (A) or pCREB1 (B) in MDA-MB-134 cells in response to E2 and FI stimulation and to DMSO treatment as the control condition.

CREB1 is recruited to endogenous ERα target genes. The ChIP experiment was performed with MDA-MB-134 cells transfected either with the wild-type or the dominant-negative forms of CREB1 before stimulation with E2 or FI.

(A, B) ERα and pCREB1 recruitment to sites that are not bound by pCREB1. ChIP experiments with MDA-MB-134 cells stimulated with E2 or FI. With this set of genes, the ERα binding site of GREB1 serves as a control of a site bound by both transcription factors. (C, D) Snapshots from the ChIP-seq data for both ERα and pCREB1 peaks are shown across indicated genes for DMSO (blue), E2 (red), and FI (green). Genomic coordinates are indicated. (E) ChIP experiments of ERα with MDA-MB-134 cells stimulated with E2 or FI, with and without CREB1 knockdown. See also Fig S8 for additional examples.

(A–D) Snapshots for unique peaks associated with genes of either (A, B) ERα or pCREB1 (C, D) from the ChIP-seq data of MDA-MB-134 cells. Genomic coordinates are indicated. This figure complements Fig S7.

ERα recruitment to its targets is stimulated by CBP, further increased by CREB1, and abolished by overexpression of the S133A mutant of CREB1. The ERα ChIP experiment with MDA-MD-134 cells was performed by transfection of plasmids for the expression of the indicated proteins and treatments as shown.

(A–C) Flow cytometry analysis of the mitochondrial membrane potential of MDA-MB-134 cells under different conditions as indicated. Cells were stained with MitoTracker Red, an indicator of the mitochondrial membrane potential. The data were acquired in the FL-2 channel, which corresponds to MitoTracker Red fluorescence and the intensities of control cells and of cells induced to undergo apoptosis with STS are shown as green and purple histograms, respectively. For each histogram, the mean value is indicated. Note that these values cannot be compared between different panels because they were obtained with different cultures of cells with different knockdowns (i.e., with control shRNA versus ERα shRNA versus siCREB1); what can be compared in each case is minus and plus induction of apoptosis (C versus STS).

(A) Both CREB1 and ERα suppress STS-induced apoptosis in response to E2 and cAMP. Knockdown of CREB1 and ERα, treatments with STS, E2, and FI, and microscopic assessment of apoptosis were performed with MDA-MB-134 cells as described in the Materials and Methods section. The data points are averages of three independent experiments obtained by the inspection of 200 cells each, and the error bars indicate the standard error of the mean. (B) High expression levels of the CREB1 gene correlate with better breast cancer outcome. The CREB1 gene was used as a marker for the Kaplan–Meier analysis of breast cancers with the GOBO tool. The plots indicate distant metastasis-free survival (DMSF) as a function of time for patients with ERα-positive (ER+) (upper panel) and ERα-negative (ER−) (lower panel) breast cancers. Within each panel, the number of samples in each category and the P-value for the difference between high and low expression cases are shown. Additional analyses are presented in Figs S11 and S12.

(A, B) Kaplan–Meier analyses of breast cancers. They are complementary to those shown in Fig 7 and were also performed with the GOBO tool and the CREB1 gene as a marker. Comparisons of samples positive or not for cancer cells in the lymph nodes (LNs), untreated or treated with tamoxifen (TAM), are shown in panels A and B, respectively.

(A–D) Interrogation of The Cancer Genome Atlas with the “Kaplan–Meier Plotter.” The numbers indicated below the panels correspond to the number of cases at the time points indicated in the graphs. Note that the numbers of breast tumors for the analyses of panels C and D were comparatively low. (E–H) Comparable sets of samples and types of analyses with the GOBO tool.