FOXA1 is a key determinant of estrogen receptor function and endocrine response

Abstract

Estrogen receptor-α (ER) is the key feature of most breast cancers and binding of ER to the genome correlates with expression of the Forkhead protein FOXA1 (also called HNF3α). Here we show that FOXA1 is a key determinant that can influence differential interactions between ER and chromatin. Almost all ER-chromatin interactions and gene expression changes depended on the presence of FOXA1 and FOXA1 influenced genome-wide chromatin accessibility. Furthermore, we found that CTCF was an upstream negative regulator of FOXA1-chromatin interactions. In estrogen-responsive breast cancer cells, the dependency on FOXA1 for tamoxifen-ER activity was absolute; in tamoxifen-resistant cells, ER binding was independent of ligand but depended on FOXA1. Expression of FOXA1 in non-breast cancer cells can alter ER binding and function. As such, FOXA1 is a major determinant of estrogen-ER activity and endocrine response in breast cancer cells.

Figure 1

Differential FOXA1 and ER binding overlaps in a cell context manner. A. Overlap in FOXA1 binding events between MCF-7, ZR75-1 and T-47D cells. B. Overlap between ER and FOXA1 in the three ER positive breast cancer cell lines. C. Relative overlap in ER and FOXA1 binding events within and between the three cell lines. The percentages represent the fraction of ER binding events in that cell line. An overlap was considered if the peaks shared at least one base pair. D. Examples of regions showing cell line specific ER and FOXA1 binding. Also shown in an example of a common region bound by ER and FOXA1 in all three cell lines. E. Average signal intensity of ER binding sites that are shared with FOXA1 binding regions or ER binding events that are not shared with FOXA1. The signal intensity of ER binding events that are not shared with FOXA1 are similar to those that overlap with FOXA1. Also included is average signal intensity for FOXA1 binding at these two separate ER binding categories.

Figure 2

ER binding to chromatin and transcriptional activity requires FOXA1. A. Western blot of cells transfected with siControl or siFOXA1. B. An example of ER binding in siControl or siFOXA1 transfected cells. C. Heatmap showing the signal intensity of ER binding in siControl or siFOXA1 transfected cells in a window of −/+ 5kb. Also shown in the signal intensity for FOXA1 at the equivalent genomic region. The heatmap represents binding events ranked from the strongest to weakest ER binding (in the siControl condition) and the adjacent columns represent the signal from the corresponding genomic region, but under the different experimental conditions. D. Smoothened scatterplot comparing ER binding intensity in siControl versus siFOXA1 transfected cells. As a control, a scatterplot representing two different siControl experiments is shown. E. Cells were transfected with siControl or siFOXA1, treated with vehicle (V) or estrogen (E) and cells were fractionated to enrich for the chromatin fraction, which was Western blotted. Histone H3 functioned as a loading control. The uncropped Western blot is in Supplementary figure 2. F. Oligonucleotide pulldown using total protein from siControl or siFOXA1 transfected cells. A double stranded biotin labelled oligonucleotide containing a perfect ERE or a mutant sequence was used and protein enriched by the oligonucleotide was Western blotted. G. Gene expression microarray analysis following transfection of siControl or siFOXA1 and treatment with vehicle or estrogen for 6hr.

Figure 3

Tamoxifen induces similar ER binding events to estrogen, in a FOXA1 dependent manner. A. Heatmap representing ER binding signal intensity in hormone deprived MCF-7 cells treated with vehicle, estrogen or tamoxifen for 45 minutes. The window represents −/+ 5kb. The heatmap represents binding events ranked from the strongest to weakest ER binding (in the estrogen condition). B. Example of ER binding under the different ligand conditions. C. Venn diagram representing the overlap in ER binding events between estrogen and tamoxifen treatment. Comparisons with published data are provided in Supplementary figure 5. D. Hormone deprived MCF-7 cells were transfected with siControl or siFOXA1 and treated with tamoxifen. ER ChIP was performed followed by real time PCR of known ER binding regions. The data are the fold enrichment over Input. The data are the average of independent replicates −/+ Std Dev. E. ER ChIP-seq binding data in hormone deprived tamoxifen resistant MCF-7 cells (Tam-R), treated with vehicle or tamoxifen. F. Tam-R cells were hormone deprived, transfected with siControl or siFOXA1 and treated with tamoxifen. Total cell growth was assessed. The data are the average of independent replicates −/+ Std Dev.

Figure 4

FOXA1 expression in U20S-ER osteosarcoma cancer cells renders ER functional. A. FOXA1 or control plasmids were transfected in U20S-ER cells and Western blotting was used to confirm expression. B. In control or FOXA1 expressing U20S-ER cells, ER ChIP was performed followed by real-time PCR of known ER binding events derived from breast cancer cells. * denotes p < 0.01. The data are the average of independent replicates −/+ Std Dev. C. U20S-ER cells were transfected with control or FOXA1 expressing vector and total chromatin fraction was collected and Western blotted for ER. Histone H3 functioned as a control. D. Oligonucleotide pulldown using total protein from control or FOXA1 transfected U20S-ER cells. A double stranded biotin labelled oligonucleotide containing a perfect ERE or a mutant sequence was used and protein enriched by the oligonucleotide was Western blotted. E. Control or FOXA1 expressing U20S-ER cells were treated with estrogen or tamoxifen and mRNA levels of known breast cancer associated target genes were assessed. The data are the average of independent replicates −/+ Std Dev. * denotes p < 0.05. Specifically for TFF1, the fold change is x10 of the y-axis values. F. Cell proliferation was performed in which cell confluence was assessed in U20S-ER cells transfected with control or FOXA1 and treated with tamoxifen. The data are the average of independent replicates −/+ Std Dev.

Figure 5

FOXA1 is required for maintaining chromatin structure. A. Genome-wide FAIRE (Formaldehyde-assisted isolation of regulatory elements) was performed in MCF-7 cells transfected with siControl or siFOXA1 and treated with vehicle or estrogen for 1hr. A. Overlap in FAIRE regions between vehicle and estrogen treatment in control cells. B. An example of FAIRE regions, some that are dependent on FOXA1 and adjacent regions that are independent of FOXA1. C. Overlap between FAIRE (both vehicle and estrogen combined) and ER binding. The different categories (ER and FAIRE positive regions versus ER but not FAIRE signal) were assessed for the fraction that represent either ER but not FOXA1 binding or shared ER and FOXA1 binding regions. Also included are the changes in FAIRE and ER binding signal within the two categories. D. Fraction of promoter proximal regions of 6hr estrogen induced or estrogen repressed genes that possess FAIRE signal. The relative difference in FAIRE signal in cells transfected with siControl or siFOXA1 is shown.

Figure 6

ER and FOXA1 shared binding events are exclusively independent of CTCF and CTCF can repress FOXA1 binding and activity. A. Overlap between FOXA1, ER and CTCF binding events in MCF-7 cells. B. Heatmap representing binding signal from regions where ER, FOXA1 and/or CTCF overlap. The categories are: I. ER and FOXA1 shared (but not CTCF) binding events, II. Regions bound by ER, FOXA1 and CTCF, III. FOXA1 and CTCF shared (but not ER) binding regions. The window represents −/+ 5kb. The FOXA1 binding events that overlap with CTCF tend to be the weakest FOXA1 binding events. C. Western blot of MCF-7 and ZR75-1 cells transfected with siControl or siCTCF. D. Heatmap showing the MCF-7 and ZR75-1 unique FOXA1 binding regions. ZR75-1 and MCF-7 cells were transfected with siControl or siCTCF. FOXA1 and H3K4me1 ChIP was performed, followed by real-time PCR of three regions that were FOXA1 binding regions exclusively in the other cell line. The data are the average of independent replicates −/+ Std Dev.