Fig. 3: ERα interacts with ZEB1 and requires AP2γ for effective ZEB1-ERα interaction and transcriptional activity. | Nature Communications

Fig. 3: ERα interacts with ZEB1 and requires AP2γ for effective ZEB1-ERα interaction and transcriptional activity.

From: Cooperative interaction between ERα and the EMT-inducer ZEB1 reprograms breast cancer cells for bone metastasis

Fig. 3

a Immunoblots of an ERα and ZEB1 co-immunoprecipitation experiment. IPs with extracts from MCF7-V-ZEB1 without or with DOX treatment (for 1 week) were performed with antibodies specific to the exogenously expressed ZEB1 (left) or to the endogenous ERα (right). A control immunoprecipitation was performed in parallel with IgG, blotted, and exposed simultaneously (results are representative of n = 3 independent experiments). b, c Venn diagrams showing overlap of AP2γ (b) or FOXA1(c) binding sites with ZEB1 and ERBSs from the ChIP-seq data. d Aggregation plot of the binding sites of ZEB1, ERα, AP2γ, FOXA1, GATA3, and P300, and the open chromatin histone marks H3K27ac, H3K4me1, and H3K9me3. Except for ERα, ChIP-seq data were from published data sets: GSE21234 (TFAP2C), GSE25315 (FOXA1), GSE60270 (GATA3, P300, H3K27ac, H3K4me1, and H3K9me3). e Genome browser snapshots of ZEB1, ERα, AP2γ, and FOXA1 enhancers of indicated genes. Highlights show shared binding sites for indicated factors. f Luciferase reporter assays with ERE-Luc in HEK293T cells infected with lentiviral constructs for shRNAs targeting FOXA1, TFAP2C, or both mRNAs; scrambled shRNA (shScr) was used as negative control (mean ± SEM, n = 3 biologically independent experiments). g Co-IPs with HEK293T cells co-transfected with ZEB1 and ERα expression vectors. A control IP was performed with an IgG antibody (results are representative of n = 3 independent experiments). h Cells infected with viruses for expression of shScr, shTFAP2C, or shFOXA1. ERα ChIP-qPCR values are represented as the fold of the shScr in −DOX (mean ± SEM, n = 4 biologically independent experiments). i–j ERα ChIP-qPCR of binding sites associated with the genes LGALS1 and RAP1GAP2 (i), and SIRT5 and CD276 (j) in MCF7-V-ZEB1 cells infected with shScr or shTFAP2C. k ChIP-qPCR of ERBS at the TFAP2C 5′-UTR (means ± SEM, n = 4 and n = 3 biologically independent experiments in i and k, and j, respectively). l Immunoblots of extracts from MCF7-V-ZEB1 cells. veh, E2, and FI stand for vehicle, 17β-estradiol, and forskolin + IBMX, respectively. For bar graphs, p values are indicated above the bars; statistical significance was determined with a two-way ANOVA. Source data are provided as a Source Data file.

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