Genome-wide mapping of FOXM1 binding reveals co-binding with oestrogen receptor alpha in breast cancer cells (ChIP-seq)

The forkhead transcription factor FOXM1 is a key regulator of the cell cycle and is overexpressed in cancer. Increased levels of FOXM1 are associated with both poor prognosis and oestrogen receptor (ERalpha) status in primary breast cancer. In this study, we map FOXM1 binding genome wide in both ERalpha-positive (MCF-7) and -negative (MDA-MB-231) breast cancer cells. We identify a common set of FOXM1 binding events at cell cycle-regulating genes, but in addition, in MCF-7 cells we find a high level of concordance with ERalpha-binding regions. FOXM1 binding at these co-binding sites is dependent on ERalpha binding, as depletion of ER protein levels reduced FOXM1 binding. FOXM1 interacts directly with both ERalpha co-activator CARM1 and is required for H3 arginine methylation at the ERalpha complex. Inhibition of FOXM1 activity with the ligand thiostrepton resulted in decreased FOXM1 binding at cca. 1400 sites genome wide and reduced expression of genes correlated with poor prognosis in ERalpha-positive tumour samples. These data demonstrate a novel role for the forkhead protein FOXM1 as an ERalpha cofactor and provide insight into the role of FOXM1 in ERalpha-positive breast cancer. The FOXM1-binding sites were mapped by ChIP-Seq in MCF-7 and MDA-MB-231 cells. Cells were treated either with thiostrepton, a FOXM1 inhibitor, or with DMSO (as control). Four replicates were performed in MCF7 cells and two replicates in MDA-MB-231 cells.

叉头框转录因子FOXM1（forkhead transcription factor FOXM1）是细胞周期的关键调控因子，在癌症中呈现过表达状态。FOXM1水平升高与原发性乳腺癌的不良预后及雌激素受体α（ERalpha，oestrogen receptor）状态密切相关。
本研究中，我们分别在雌激素受体α阳性（ERalpha-positive，MCF-7）与阴性（ERalpha-negative，MDA-MB-231）乳腺癌细胞内，完成了FOXM1的全基因组结合位点图谱绘制。我们鉴定出一组位于细胞周期调控基因上的FOXM1共有结合事件；不仅如此，在MCF-7细胞中，FOXM1结合区域与ERalpha结合区域存在高度重合。
FOXM1在上述共结合位点的结合依赖于ERalpha的结合：敲低ER蛋白水平可显著削弱FOXM1的结合能力。FOXM1可直接与ERalpha共激活因子CARM1发生相互作用，且对于ERalpha复合物处的H3精氨酸甲基化过程不可或缺。
使用配体硫链丝菌素（thiostrepton）抑制FOXM1活性后，全基因组范围内约1400个位点的FOXM1结合水平显著降低，同时ERalpha阳性肿瘤样本中与不良预后相关的基因表达量也出现下调。上述数据揭示了叉头框蛋白FOXM1作为ERalpha共因子的全新功能，为阐明FOXM1在ERalpha阳性乳腺癌中的作用提供了新的研究视角。
本研究通过染色质免疫共沉淀测序（ChIP-Seq）在MCF-7与MDA-MB-231细胞中绘制了FOXM1的结合位点图谱：实验细胞分别经FOXM1抑制剂硫链丝菌素处理，或以二甲基亚砜（DMSO）处理作为空白对照。MCF-7细胞组设置了4次生物学重复，MDA-MB-231细胞组则设置了2次生物学重复。