Characterization of the mechanism of phytoestrogen responsive gene expression at chromatin level in MCF-7 breast cancer cells

Phytoestrogens (PEs) are naturally occurring plant derived non-steroidal hormonal compounds present in our diet. They are structurally similar to estrogens and function by binding to the promoter region of various genes and regulate their expression. The regulation of gene expression occurs at different levels including the direct DNA-protein interaction, translation of messenger RNA, and remodeling of chromatin. A plethora of evidence demonstrates that phytoestrogen (PE) upregulates gene expression through activation of the estrogen responsive element (ERE) and activator protein 1 (AP-1) regulatory elements present in estrogen responsive genes. However, there is no report showing the application of chromatin immunoprecipitation (ChIP) assay in studying the interaction of PE-estrogen receptor (ER) complex with the promoter regions of these genes. Therefore, the overall goal of this study was to characterize the mechanism of action of PEs in regulating gene expression at the level of chromatin. Since chromatin regulation is central to PE action, the specific aims of the study were: (1) evaluate chromatin involvement in the regulation of PE responsive genes Guanine Nucleotide Binding protein 1 (GNB1), Progesterone receptor (PgR) and Trefoil factor (pS2). In addition, the involvement of ER in PE regulation at chromatin level will be determined, (2) to demonstrate that the transactivation of these PE responsive genes involves direct DNA binding by PE-ER complex.;In this report, it was confirmed that GNB1 is a novel PE regulated gene. Northern blot and quantitative real time-polymerase chain reaction demonstrated that pS2 and PgR are upregulated at the transcriptional level. Furthermore, it was shown that the transcriptional regulation of these genes also took place through direct DNA binding at chromatin level in a phytoestrogen dependent manner. The ChIP analysis of PE responsive genes indicated that GNB1, PgR and pS2 require ER for gene regulation. In case of the PgR gene, the ChIP analysis suggested that the PE-ER complex was not involved in chromatin regulation. Guanine nucleotide binding protein beta subunit 1 promoter DNA sequence revealed that the promoter contains several putative ERE half sites. Direct binding of ERalpha to the GNB1 promoter was confirmed by an electrophoretic mobility shift analysis. Furthermore, exposure of MCF-7 breast carcinoma cells to genistein (GE) induced an increase in the RNA level of GNB1 gene. The transactivation was confirmed by showing an increase in luciferase activity when pGL3P (GNB1 -C9) was transfected into MCF-7 breast carcinoma cells following induction with GE. This data confirmed that the gene expression of GNB1 was regulated by the ERE and specificity protein-1 (SP1) sites present in its promoter. This study for the firsttime demonstrated the direct regulation of two PE responsive genes at the chromatin level.