Estrogen receptor α (ERα), FOXA1, and GATA3 form a functional enhanceosome in MCF-7 breast carcinoma cell that is significantly associated with active transcriptional features such as enhanced p300 co-activator and RNA Pol II recruitment as well as chromatin opening.The enhanceosome exerts significant impact and optimal transcriptional control in the regulation of E2-responsive genes.The presence of FOXA1 and GATA3 is indispensable in restoring the ERα growth-response machinery in the ERα-negative cells and recapitulating the appropriate expression cassette.
Estrogen receptor α (ERα) is a ligand-inducible hormone nuclear receptor that has important physiology and pathology roles in reproduction, cancer, and cardiovascular biology. The regulation of ERα involves its binding to the DNA recognition sequence also known as estrogen-response elements (EREs) and recruits a variety of co-activators, corepressors, and chromatin remodeling enzymes to initiate transcription machinery. In our previous (Lin et al, 2007) and recent (Joseph et al, 2010) studies, we have identified high confidence ERα binding sites in MCF-7 human mammary carcinoma cells. With known motif scanning and de novo motif detection, we identified that FOXA1 and GATA3 motifs were commonly enriched around ERα binding sites. Moreover, numerous microarray studies have documented the co-expression of ERα, FOXA1, and GATA3 in primary breast tumors (Badve et al, 2007; Wilson and Giguere, 2008). This evidence suggests that these three transcription factors (TFs) may cluster on DNA binding sites and contribute to the breast cancer phenotype. However, there is little understanding as to the nature of their coordinated interaction at the genome level or the biological consequences of their detailed interaction.
We mapped the genome-wide binding profiles of ERα, FOXA1, and GATA3 using the massive parallel chromatin immunoprecipitation-sequencing (ChIP-seq) approach. We observed that ERα, FOXA1, and GATA3 colocalized in a coordinated manner where ∼30% of all ERα binding sites were overlapped with FOXA1 and GATA3 bindings upon estrogen (E2) stimulation. Moreover, we found that the ERα+FOXA1+GATA3 conjoint sites were associated with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Such results indicate that these three TFs form a functional enhanceosome and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. And such enhanceosome binding sites appear to regulate the genes driving core ERα function.
To further validate that ERα+FOXA1+GATA3 co-binding represents an optimal configuration for E2-mediated transcriptional activation, we have performed luciferase reporter assays on GREB1 locus that actively engages ERα enhanceosome sites in gene regulation (Figure 5C). The presence of ERα induced the GREB1 luciferase activity to ∼246% (as compared with the control construct). The individual presence of FOXA1 and GATA3 or combination of both only produced subtle changes to the GREB1 luciferase activity. The combination of ERα+FOXA1 and ERα+GATA3 has increased the luciferase activity to ∼330%. Interestingly, the assemblage of ERα+FOXA1+GATA3 provided the optimal ER responsiveness to 370%. This suggests that ERα provides the fundamental gene regulatory module but that FOXA1 and GATA3 incrementally improve ERα-regulated transcriptional induction.
It is known that ERα is a ligand-activated TF that mediates the proliferative effects of E2 in breast cancer cells. Garcia et al (1992) showed inhibited growth in MDA-MB-231 cells with forced expression of ERα upon E2 treatment. The rationale for these different outcomes has remained elusive. We posited that these higher order regulatory mechanisms of ERα function such as the formation and composition of enhanceosomes may explain the establishment of transcriptional regulatory cassettes favoring either growth enhancement or growth repression.
To test this hypothesis, we stably transfected the MDA-MB-231 cells with individual ERα, FOXA1, GATA3, or in combinations (Figure 6A). We observed inhibited growth in cells with enforced expression of ERα or FOXA1. There was unaltered growth in cells with expression of GATA3. Co-expression of ERα+FOXA1 or ERα+GATA3 exhibited inhibition of cell proliferation as compared with control cells. However, the co-expression of ERα together with FOXA1 and GATA3 resulted in marked induction of cell proliferation under E2 stimulation. We have recapitulated this cellular reprogramming in another ERα-negative breast cancer cell line, BT-549 and observed similar E2-responsive growth induction in the ERα+FOXA1+GATA3-expressing BT-549 cells. This suggests that only with the full activation of conjoint binding sites by the three TFs will the proliferative phenotype associated with ligand induced ERα be manifest.
To assess the nature of this transcriptional reprogramming, we asked the question if the reprogrammed MDA-MB-231 cells display any similarity in the expression profile of the ERα-positive breast cancer cell line, MCF-7 (Figure 6C). We combined the E2-regulated genes from these differently transfected MDA-MB-231 cells, and compared their expressions in these MDA-MB-231-transfected cells and MCF-7 cells. Strikingly, we found that the expression profiles of ERα+FOXA1+GATA3-expressing MDA-MB-231 cells display a good correlation (R=0.42) with the E2-induced expression profile of MCF-7. We did not observe such correlation between the expression profiles of MDA-MB-231 transfected with ERα only (R=−0.21). Furthermore, we observed that there is marginal induced expression of luminal marker genes and reduced expression of basal genes in the ERα+FOXA1+GATA3-expressing MDA-MB-231 as compared with the vector control cells. This suggests that the enhanceosome component is competent to partially reprogramme the basal cells to resemble the luminal cells.
Taken together, we have uncovered the genomics impact as well as the functional importance of an enhanceosome comprising ERα, FOXA1, and GATA3 in the estrogen responsiveness of ERα-positive breast cancer cells. This enhanceosome exerts significant combinatorial control of the transcriptional network regulating growth and proliferation of ERα-positive breast cancer cells. Most importantly, we show that the transfection of the enhanceosome component was necessary to reprogramme the ERα-negative cells to restore the estrogen-responsive growth and to transcriptionally induce a basal to luminal transition.
Despite the role of the estrogen receptor α (ERα) pathway as a key growth driver for breast cells, the phenotypic consequence of exogenous introduction of ERα into ERα-negative cells paradoxically has been growth inhibition. We mapped the binding profiles of ERα and its interacting transcription factors (TFs), FOXA1 and GATA3 in MCF-7 breast carcinoma cells, and observed that these three TFs form a functional enhanceosome that regulates the genes driving core ERα function and cooperatively modulate the transcriptional networks previously ascribed to ERα alone. We demonstrate that these enhanceosome occupied sites are associated with optimal enhancer characteristics with highest p300 co-activator recruitment, RNA Pol II occupancy, and chromatin opening. Most importantly, we show that the transfection of all three TFs was necessary to reprogramme the ERα-negative MDA-MB-231 and BT-549 cells to restore the estrogen-responsive growth resembling estrogen-treated ERα-positive MCF-7 cells. Cumulatively, these results suggest that all the enhanceosome components comprising ERα, FOXA1, and GATA3 are necessary for the full repertoire of cancer-associated effects of the ERα.