Comprehensive Genomic Analysis Reveals that the Pioneering Function of FOXA1 Is Independent of Hormonal Signaling

Abstract

Considerable work has linked hormone receptors, such as estrogen receptor-alpha (ER), with the pioneer factor FOXA1. Altered FOXA1 levels contribute to endocrine-resistant breast cancer, where it maintains ER-chromatin interactions, even in contexts in which cells are refractory to ER-targeted drugs. A recent study controversially suggests that FOXA1 binding can be induced by hormonal pathways, including the estrogen-ER complex. We now show that the vast majority (>99%) of FOXA1 binding events are unaffected by steroid activation. A small number (<1%) of FOXA1 binding sites appear to be induced by estrogen, but these are created from chromatin interactions between ER binding sites and adjacent FOXA1 binding sites and do not represent genuine new FOXA1-pioneering elements. FOXA1 is therefore not regulated by estrogen and remains a bone fide pioneer factor that is entirely upstream of the ER complex.

Graphical abstract

Figure 1

PCA and Unbiased Clustering of the Different ChIP-Seq Datasets Read densities from aligned libraries of equal size of 20 million reads were measured on corresponding FOXA1 binding sites from Swinstead et al. (2016) (GEO: GSE72249). (A) The peaks for all treatments were merged in a single set prior to the measurement for each study, and obtained data were subjected to PCA. The PCA plots illustrate degree of similarity between the replicates. Spearman rank correlation between ER-mediated chromatin interactions (ChIA-PET) and the 357 estrogen-induced FOXA1 binding sites (ab5089). (B) Hierarchical clustering of the Swinstead et al. (2016) binding sites. For hierarchical clustering of the Swinstead et al. (2016) binding sites, the yielded read densities were normalized using median absolute deviation and clustered in MATLAB framework using the “ward” method with the linkage function. The duplicate samples from Swinstead et al. (2016) did not cluster on the basis of treatment condition. (C) PCA of our FOXA1 ChIP-seq generated with two different FOXA1 antibodies (ab23738 and ab5089). (D) Hierarchical clustering of our FOXA1 binding sites, showing clustering on the basis of replicates.

Figure 2

Analysis of FOXA1 ChIP-Seq Binding with Two Separate Antibodies in Response to Estrogen Treatment in MCF-7 and ZR-75-1 Cells (A, C, D, and F) ChIP-seq tag densities visualized at FOXA1-occupied genomic locations in control and estrogen-treated MCF-7 (A and C) and ZR-75-1 (D and F) cells, using antibodies ab23738 and ab5089. (B and E) Zoomed heatmap shows differential binding of FOXA1 specific to ab5089 in MCF-7 cells (B) and ZR-75-1 (E), respectively. (G) Overlap of estrogen-enriched FOXA1 binding sites between MCF-7 and ZR-75-1 cells. (H) Transcription factor motifs found overrepresented in the common and estrogen-induced FOXA1 sites.

Figure 3

Integration of the Estrogen-Enriched FOXA1 Binding Events with Estrogen-Mediated Gene Expression Events (A) RNA sequencing (RNA-seq) expression profile following short-term (3 h) estrogen treatment of MCF-7, shown as a dispersion plot. (B) Gene set enrichment analysis (GSEA) pre-ranked test correlating estrogen-induced genes with the 357 estrogen-induced FOXA1 binding sites. (C) Examples of sites co-bound by FOXA1 and ER, as well as sites unique to each of the two transcription factors. (D and E) Proximity of estrogen-induced FOXA1 peaks and the closest ER (D) or FOXA1 (E) site. Heatmap represents FOXA1-gained sites in red.

Figure 4

ER Binding Mediates Indirect FOXA1 Binding via Chromatin Looping at cis-Regulatory Elements (A) Correlation between ER-mediated chromatin interactions (ChIA-PET) and the 357 estrogen-induced FOXA1 binding sites (ab5089). The table shows the correlation values between ChIA-PET interactions and the 357 estrogen-induced FOXA1 binding sites. (B) Examples of ER and FOXA1 peaks at regions that are involved in chromatin loops, as detected by ChIA-PET. The images of the ChIA-PET loops are taken from Fullwood et al. (2009).