Figure 3

FOXM1 binding at ERα co-bound sites is dependent on ERα. (a) FOXM1 binding was assessed following depletion of ER by fulvestrant (10 nM) treatment for 3 h. Western blot showing depletion of ERα whilst FOXM1 protein levels are unchanged. (b) ChIP for FOXM1 was followed by qPCR to assess binding at ERα co-bound sites and FOXM1-only binding sites. ERα binding was assessed following depletion of FOXM1 by siRNA treatment for 48 h. (c) Western blot showing depletion of FOXM1 whilst ERα protein levels are not significantly changed. (d) ChIP for ER followed by qPCR to detect binding at FOXM1 co-bound regions. Depletion of FOXM1 affects ERα-regulated gene expression. (e) qPCR of ERα-regulated gene expression in MCF7 cells treated with siRNA for siControl or FOXM1 for 48 h. Nascent and total mRNA levels were measured. FOXM1 interacts directly with the co-activator CARM1 and regulates CARM1-mediated histone H3 arginine methylation. (f) Co-immunoprecipitation showing pull-down of CARM1 with FOXM1 antibody using either low binding (LB) or high binding (HB) immunoprecipitation buffers additionally supplemented with dithiothreitol (DTT). (g) ChIP for methylated arginine 17 on histone H3 followed by qPCR for regions of FOXM1 and ER co-binding. Data are normalized to total H3 in MCF7 transfected with siRNA for 48 h (siControl or FOXM1). (h) Proposed simplified model for FOXM1/ER/CARM1 complex in transcription regulation at enhancer regions. Data representative of triplicate experiments ± standard deviation. *P < 0.05, **P < 0.01, ***P < 0.001.