GATA3 acts upstream of FOXA1 in mediating ESR1 binding by shaping enhancer accessibility

Abstract

Estrogen receptor (ESR1) drives growth in the majority of human breast cancers by binding to regulatory elements and inducing transcription events that promote tumor growth. Differences in enhancer occupancy by ESR1 contribute to the diverse expression profiles and clinical outcome observed in breast cancer patients. GATA3 is an ESR1-cooperating transcription factor mutated in breast tumors; however, its genomic properties are not fully defined. In order to investigate the composition of enhancers involved in estrogen-induced transcription and the potential role of GATA3, we performed extensive ChIP-sequencing in unstimulated breast cancer cells and following estrogen treatment. We find that GATA3 is pivotal in mediating enhancer accessibility at regulatory regions involved in ESR1-mediated transcription. GATA3 silencing resulted in a global redistribution of cofactors and active histone marks prior to estrogen stimulation. These global genomic changes altered the ESR1-binding profile that subsequently occurred following estrogen, with events exhibiting both loss and gain in binding affinity, implying a GATA3-mediated redistribution of ESR1 binding. The GATA3-mediated redistributed ESR1 profile correlated with changes in gene expression, suggestive of its functionality. Chromatin loops at the TFF locus involving ESR1-bound enhancers occurred independently of ESR1 when GATA3 was silenced, indicating that GATA3, when present on the chromatin, may serve as a licensing factor for estrogen-ESR1-mediated interactions between cis-regulatory elements. Together, these experiments suggest that GATA3 directly impacts ESR1 enhancer accessibility, and may potentially explain the contribution of mutant-GATA3 in the heterogeneity of ESR1+ breast cancer.

Figure 1.

Silencing of GATA3 results in redistribution of ESR1 binding globally. (A) GATA3 binding at the TFF1 locus in unstimulated and E2-treated MCF7 cells. (B) Venn diagram showing overlap in ESR1, FOXA1, and GATA3 binding in MCF7 cells. (C) MA plot of Differential Binding Affinity (DBA). Analysis using edgeR of ESR1 binding in siControl versus siGATA3 conditions of three biological replicates. The universe of ESR1-binding events consists of 49,626 peaks, which were used in the DBA analysis. The x-axis shows the log concentration of sequenced tags per peak. The y-axis represents the log fold change of siControl over siGATA3. A third of all ESR1-binding events showed significant changes in affinity at FDR < 0.1. The binding events plotted red above 0 on the y-axis represent Weaker ESR1 binding in siGATA3 (16%), whereas the red at the bottom represents events with Stronger ESR1 affinity in siGATA3 (17%). (D) A representative example of the redistributed ESR1 binding in siGATA3 MCF7 cells. (E, left) Heatmaps of the ChIP-seq data sets centered on the ESR1 peaks and their averaged signal intensity (right) show that the Weaker ESR1 events are cobound by GATA3, suggesting that at these regions GATA3 mediates ESR1–chromatin associations. However, GATA3 binding did not overlap at regions where ESR1 binding was gained following silencing of GATA3.

Figure 2.

GATA3-dependent ESR1-binding events show distinct transcription factor co-occupancy and histone modifications. (A) A binary co-occupancy analysis showing the percentage of the ESR1 categories affected by depletion of GATA3 normally co-occupied by GATA3 and/or FOXA1. GATA3 and FOXA1 binding is from wild-type MCF7 cells. (Data from Hurtado et al. 2011.) (B) Averaged signal intensity of H3K4me1 and H3K27Ac active histone marks in unstimulated (Veh) and estrogen (E2)-treated cells centered on the different categories of ESR1 binding influenced by silencing of GATA3. (C) De novo motif analysis (Weeder).

Figure 3.

Silencing GATA3 modulates enhancer accessibility by redirecting FOXA1, EP300, H3K4me1, and H3K27Ac histone marks prior to ESR1-E2 recruitment. (A) FOXA1, EP300, H3K4me1, and H3K27Ac were mapped by ChIP-seq, following silencing of GATA3 or siControl. Silencing of GATA3 results in an altered histone profile at enhancer elements prior to ESR1 recruitment (Veh) at the same regions that will harbor the redistributed ESR1 binding upon E2 stimulation. (B) An example of the changes in the histone landscape and EP300 recruitment at TFF3, an E2 up-regulated gene. Silencing of GATA3 results in a gain of H3K4me1, EP300, and subsequent H3K27Ac in unstimulated cells, which parallels the E2-dependent gain in ESR1-binding affinity. (C) Average ChIP-seq signal intensity of FOXA1 and EP300 binding in unstimulated (Veh) MCF7-siGATA3 and siControl cells, centered on the redistributed ESR1-siGATA3 events. (D) Average ChIP-seq signal intensity of H3K4me1 and K3K27Ac binding in unstimulated (Veh) MCF7-siGATA3 and siControl cells, centered on the redistributed ESR1–siGATA3 events.

Figure 4.

MCF7 cells depleted of GATA3 have an altered E2 transcriptional program that correlates with the redistributed ESR1 binding. (A) Integration of the Differentially Expressed Genes (DEG) with the redistributed ESR1 ChIP-seq data sets. A 50-kb window around the transcription start sites (TSS) of all DEG targets was overlapped with the altered ESR1 binding in siGATA3 conditions. ESR1 stronger binding is enriched near genes that are overexpressed in siGATA3 compared with siControl (bottom of gene cluster I, cluster III, and cluster IV). ESR1 Weaker events are in the vicinity of genes down-regulated by siGATA3 (Gene clusters 1 and II). Also included (right) is information showing whether each gene has an ESR1-binding event within 50 kb of the TSS. Included is ESR1 binding that is gained (green), lost (yellow), or not changed (blue) following GATA3 silencing. (B) Gene Set Analysis Enrichment (GSEA) reveals that ESR1 Stronger events after GATA3 silencing are enriched near up-regulated genes, whereas ESR1–Weaker siGATA3-binding events correlate with transcriptional down-regulation in siGATA3-treated cells.

Figure 5.

Redistribution of ESR1 binding after GATA3 silencing correlates with altered gene transcription. Weaker ESR1–siGATA3 binding correlates with decreased gene expression at the (A) CA12, (B) CAV1, and (C) METRNL genes. Stronger ESR1–siGATA3 binding correlates with increased gene transcription of the (D) TRAK1, (E) TGFB1, and (F) FGFR3 genes. (Left) Snapshots of ESR1 binding in siControl and siGATA3 estrogen conditions; (right) log2 quantile normalized averaged intensities of six biological replicates with the corresponding Illumina probes. For the METRNL gene we omitted the probes ILMN_2342066 and ILMN_2258004 due to space restrictions; however, they agreed with the other probes.