From the Cover: Location analysis of estrogen receptor alpha target promoters reveals that FOXA1 defines a domain of the estrogen response

Abstract

Nuclear receptors can activate diverse biological pathways within a target cell in response to their cognate ligands, but how this compartmentalization is achieved at the level of gene regulation is poorly understood. We used a genome-wide analysis of promoter occupancy by the estrogen receptor alpha (ERalpha) in MCF-7 cells to investigate the molecular mechanisms underlying the action of 17beta-estradiol (E2) in controlling the growth of breast cancer cells. We identified 153 promoters bound by ERalpha in the presence of E2. Motif-finding algorithms demonstrated that the estrogen response element (ERE) is the most common motif present in these promoters whereas conventional chromatin immunoprecipitation assays showed E2-modulated recruitment of coactivator AIB1 and RNA polymerase II at these loci. The promoters were linked to known ERalpha targets but also to many genes not directly associated with the estrogenic response, including the transcriptional factor FOXA1, whose expression correlates with the presence of ERalpha in breast tumors. We found that ablation of FOXA1 expression in MCF-7 cells suppressed ERalpha binding to the prototypic TFF1 promoter (which contains a FOXA1 binding site), hindered the induction of TFF1 expression by E2, and prevented hormone-induced reentry into the cell cycle. Taken together, these results define a paradigm for estrogen action in breast cancer cells and suggest that regulation of gene expression by nuclear receptors can be compartmentalized into unique transcriptional domains by means of licensing of their activity to cofactors such as FOXA1.

Fig. 1.

Genome-wide location analysis of direct ERα transcriptional targets in MCF-7 breast cancer cells. (A) Motif-finding algorithms identify the consensus ERE (GGTCANNNTGACCT) as the most common transcription factor-binding motif present in the promoters bound by ERα in the promoter array. The motif was present in 60% of the promoters used for the analysis. (B) E₂-modulated recruitment of ERα, the coactivator AIB1, and RNA polymerase II at selected ERα targets in MCF-7 cells as assayed by conventional ChIP.

Fig. 2.

FOXA1, a target of ERα, is recruited to a subset of ERα targets. (A) Pie chart representing major biological functions and processes associated with ERα targets (153) enriched in E₂-treated MCF-7 cells. (B) Induction of FOXA1 expression by E₂ as monitored by Western blot. (C) FOXA1 recruitment to a subset of ERα-bound promoters containing FOXA1 binding sites as assayed by conventional ChIP. The STS and HK1 promoters serve as a ERα-bound control promoters without a FOXA1-binding site. The results presented are from a single experiment representative of three independent experiments.

Fig. 3.

FOXA1 is required for ERα activity on a subset of target promoters. (A) FOXA1 expression in MCF-7 cells transfected with control (siC) and FOXA1 (siF) siRNAs. Actin levels serve as a control for specificity and gel loading. (B) FOXA1 is required for the E₂ regulation of TFF1 expression in MCF-7 cells. RT-PCR analysis of TFF1 expression was performed with extracts obtained from cells transfected with control (siC) and FOXA1 (siF) siRNAs in the presence or absence of E₂. The STS promoter serves as an ERα-bound control promoter without a FOXA1 binding site. (C) Knock-down of FOXA1 expression decreases the ability of ERα to stimulate transcription from the TFF1 promoter. MCF-7 cells were cotransfected with ERα, the TFF1-Luc reporter, and control (siC) or FOXA1 (siF) siRNAs in the presence or absence of E₂.(D) FOXA1 is required for E₂-induced recruitment of ERα to the TFF1, RPS6KL1, ABCC5, and UGT2B17 promoters as assayed by conventional ChIP. The STS promoter acts as a control as described in B. The cells were treated with vehicle (C) or 100 nM E₂. Results are expressed as the percentage of maximal ERα binding observed in the presence of E₂. For panel A, B and C, the results presented are from a single experiment representative of at least two independent experiments.

Fig. 4.

Effect of FOXA1 knock-down on cell cycle entry in response to E₂. Results shown represent the percentage change in cells in S, G₂, and M phases stimulated by E₂ relative to untreated cells in the presence of control (siC) or FOXA1 (siF) siRNAs. The results presented are from a single experiment representative of two independent experiments.

Fig. 5.

Model illustrating how FOXA1 licensing defines subdomains of E₂ action in breast cancer cells. Green arrows represent direct transcriptional activity of ERα and FOXA1, and the dashed blue arrow indicates the action of FOXA1 as a modulator of ERα binding to a subset of promoters. The presence of FOXA1 thus grants permission to ERα to regulate a subset of the hormonal response, which can be further amplified by positive regulation of FOXA1 expression by E₂-bound ERα.