Figure 2

FOXM1 is elevated by tamoxifen treatment and genome-wide analysis of FOXM1, ERK2 and ERα chromatin binding sites by ChIP-seq after tamoxifen treatment, and gene expression profiling, and clustering analyses. (A) FOXM1 protein levels in MCF-7 and tamoxifen-resistant (TamR) cells monitored by Western blot analysis. (B) mRNA levels of FOXM1 mRNA over time of 1 μM 4-hydroxytamoxifen (OH-TAM) exposure. The fold change in FOXM1 gene expression in the presence of tamoxifen- over vehicle-treated cells was calculated using the comparative threshold cycle method, with the ribosomal protein 36B4 mRNA used as an internal control. (C) Proliferation of TamR control cells (Ctrl) and cells with FOXM1 knockdown (siFOXM1). OD, Optical density. Data are mean ± SEM. **P < 0.01. (D) Heat map representing the expression levels of OH-Tam-regulated genes in Ctrl and siFOXM1 MCF-7 cells treated with control vehicle or OH-Tam. Heat map shows fold change for gene expression in Tam-treated vs. vehicle-treated cells with or without siFOXM1. (E) Venn diagram showing overlap of FOXM1, extracellular signal-regulated kinase 2 (ERK2) and estrogen receptor α (ERα) chromatin binding sites in cells after 45 minutes of OH-Tam treatment and chromatin immunoprecipitation followed by high-throughput sequencing (ChIP-seq) analysis. FOXM1-Tam and ERK2-Tam cistrome data are from this study; the ERα-Tam cistrome data are derived from Hurtado et al. [31]. (F) Clustering of the binding sites for FOXM1, ERK2 and ERα after cell treatment with Tam using seqMINER software based on co-occupancy of the different factors within a 600-bp window. (G) Conservation of clusters C1, C2, C3 and C4 binding sites among vertebrates. (H) Genomic location of clusters C1 to C4 binding sites identified by using the web-based CEAS tool. UTR, Untranslated region.