Transducin-like enhancer protein 1 mediates estrogen receptor binding and transcriptional activity in breast cancer cells

Abstract

Estrogen receptor (ER) binds to distal enhancers within the genome and requires additional factors, such as the Forkhead protein FoxA1, for mediating chromatin interactions. We now show that the human Groucho protein, Transducin-like enhancer protein 1 (TLE1), positively assists some ER-chromatin interactions, a role that is distinct from its general role as a transcriptional repressor. We show that specific silencing of TLE1 inhibits the ability of ER to bind to a subset of ER binding sites within the genome, a phenomenon that results in perturbations in phospho-RNA Pol II recruitment. Furthermore, TLE1 is essential for effective ER-mediated cell division. We have discovered a distinct role for TLE1, as a necessary transcriptional component of the ER complex, where it facilitates ER-chromatin interactions.

Fig. 1.

TLE1 binds to chromatin at ER binding events and is not influenced by estrogen. (A) TLE1 ChIP after vehicle (white bars) or estrogen (black bars) treatment followed by real-time PCR of ER binding sites. TLE1 binds to a subset of regions tested independently of ligand. The data are the average of three replicate experiments ± Std Dev. (B) siRNA to TLE1 (or control siRNA) was transfected into cells and total protein was immunoblotted. The uncropped figure is in Fig. S1. C. siControl or siTLE1 transfected MCF-7 cells were treated with estrogen for 24 h and %S phase was determined by PI staining and flow cytometry. The %S phase was decreased when TLE1 was silenced. The data are the average of three independent replicates ± Std Dev and example histograms are shown in Fig. S1.

Fig. 2.

Silencing of TLE1 inhibits a subset of ER binding sites. (A) Cells were transfected with control or TLE1 siRNA and subsequently treated with estrogen for 45 min. ER ChIP-sequencing was performed. The data represent the overlap in ER binding peaks in the presence and absence of TLE1 from two independent replicates. We compared the ER binding signal intensity in the two categories (the TLE1-independent and TLE1-dependent ER binding events). The signal intensity is shown as an average of all the binding events in that category, in a window of ± 1 kb from the center of the binding event. (B) An example of an ER binding event that is lost upon silencing TLE1 (TLE1-dependent) and one example where ER binding remains (TLE1-independent).

Fig. 3.

TLE1 dependent ER binding events are less likely to be cobound by FoxA1. (A) Heat map showing ER binding signal intensity for each individual ER ChIP-seq replicate following transfection of siTLE1 or control siRNA. The heat map represents binding signal intensity with binding events ranked from the strongest to the weakest binding events. Each line represents an individual binding event, and the data are shown in a window of ± 5 kb around the center of the binding event. Also shown is the FoxA1 binding signal intensity in each category at the corresponding region and the fraction of each category (TLE1-independent or TLE1-dependent ER binding events) that overlap with a FoxA1 binding event. (B) Hormone-deprived MCF-7 cells were transfected with siRNA to TLE1 (or control siRNA) or FoxA1. ChIP of the reciprocal factor was performed followed by real-time PCR of known ER binding events. FoxA1 binding was not influenced by silencing of TLE1, but TLE1 binding was decreased when FoxA1 was silenced. N/S denotes not significant and * denotes p < 0.05. (C) Changes in mRNA levels of FoxA1 or TLE1 when the other gene was silenced by siRNA.

Fig. 4.

ER-mediated gene expression requires TLE1. Cells were hormone deprived, transfected with siControl or siTLE1, treated with estrogen for 1 h and phospho-RNA Pol II ChIP-seq was performed. We considered differentially regulated genes those that changed by more that 1.5-fold (between siTLE1 and siControl treatments) in both independent replicates. (A) An example of an estrogen-regulated gene (CTSD) and the changes in phospho-RNA Pol II occupancy and ER binding in the presence (siControl) or absence (siTLE1) of TLE1. Following silencing of TLE1, ER binding, and phospho-RNA Pol II occupancy is decreased. (B) The 312 genes contained reproducible changes in phospho-RNA Pol II occupancy following silencing of TLE1, of which 304 are genes that show decreased phospho-RNA Pol II occupancy in the absence of TLE1. Also shown is the phospho-RNA PolII binding intensity around the transcription start sites of the TLE1 dependent genes, in a window of ± 5 kb. Phospho-RNA Pol lI occupancy is decreased following silencing of TLE1. (C) Several genes were assessed using quantitative RT-PCR following silencing of TLE1 in proliferating cells. Also included are changes in TLE1, ESR1 (ER) and FoxA1 mRNA levels as controls. * denotes p < 0.05 and ** denotes p < 0.01. (D) The 304 TLE1 dependent genes were analyzed for enriched biological pathways. We found the gene signatures for the ER pathway to be overrepresented. (E) FAIRE was conducted in MCF-7 cells transfected with siControl or siTLE1. Real-time PCR of several ER binding region were assessed. The experiment was repeated in quadruplicate and one representative experiment is shown.