Fig 2: CCNB1 is transcriptionally activated by FOXM1. (A) The transcription factors for CCNB1 predicted by the TRRUST website; (B) correlation between FOXM1 and CCNB1 expression in TCGA-CESC dataset analyzed by Spearman’s rho correlation; (C) the binding relationship of FOXM1 to the CCNB1 promoter predicted by the JASPAR website; (D) the conservative binding sequence of FOXM1; (E) binding of FOXM1 to the CCNB1 promoter in Caski and Siha cells detected by ChIP-qPCR experiments; (F) luciferase activity in 293T cells co-transfected with the FOXM1 overexpression plasmid and the luciferase reporter vector assessed by luciferase reporter assay; (G) the expression of FOXM1 in normal cervical tissue and CESC predicted by GEPIA website; (H) the mRNA expression of FOXM1 in normal cervical epithelial cells HaCaT and in CSCC cells measured by RT-qPCR; (I) the expression of FOXM1 in normal cervical epithelial cells HaCaT and in CSCC cells measured by Western blot. The experiments were performed in triplicate and expressed as mean ± SD. One-way analysis of variance followed by Tukey’s test were applied for statistical analysis (E, F, H and I). *p < 0.05 vs normal tissues; **p < 0.01 vs HaCaT cells, IgG or oe-NC treatment.

Fig 3: SETD3 interacts with and methylates FoxM1 in cells.(A) FoxM1 was immunoprecipitated from cells using an anti-FoxM1 antibody or control beads followed by western blot analysis with the indicated antibodies. Input is shown in the bottom panel. (B,C) HEK-293T cells were transfected with the indicated plasmids or with an empty plasmid. The chromatin fraction was then subjected to immunoprecipitation with FLAG M2 beads followed by western blot with the indicated antibodies. (D) Cells were transfected without or with HA-SETD3 and whole cell lysate was subjected to immunoprecipitation with a pan-methyl antibody followed by western blot with the indicated antibodies. (E) Chromatin isolated fraction of the transfected cells were subjected to immunoprecipitation with FoxM1 antibody followed by immunoprecipitation with pan-methyl antibody.

Fig 4: Mutation of the FOXM1 DBD inhibits DNA binding. a Sequence alignments of the DBD for a number of Forkhead family members with the secondary structure shown schematically above. The residues used to generate point mutations are outlined in red. (*) conserved amino acids. H1-3 are a-helices, the orange arrows are ß strands, and W1-2 are winged domains. b Plot showing relative change of polarization of a fluorescently-labeled (6FAM) dsDNA FKH consensus oligonucleotide upon addition of increasing concentrations of GST-FOXM1 WT or mutant DBD proteins. The FP assay provides a quantitative method and non-disruptive method to determine FOXM1 affinity for target by measuring the fluorescence polarization signals from the FAM-labeled FKH consensus (see Materials and methods). Data are plotted as % binding and show mean ± SD of triplicate experiments. (WT Kd = 1.10 ± 0.02 µM, H287A Kd = 3.04 ± 0.10 µM). c Plots showing relative luciferase activity of a 6X DB-TATA-luciferase reporter in cells transiently transfected with either WT or DBD mutant FOXM1 with the T596A mutation as a positive control. Data are shown as fold induction of luciferase activity following doxycycline induction. d Plot showing fold induction of a luciferase reporter containing a 200 bp sequence taken from the CCNB1 promoter following doxycycline induction of WT and mutant FOXM1 expression. Data represent triplicate experiments ± SD. (*) P <0.05, (**) P <0.01, (***) P <0.001

Fig 5: GFP-tagged FOXM1 shows a similar genomic distribution to endogenous FOXM1. a Venn diagram showing the overlap between endogenous FOXM1 and GFP-FOXM1 binding sites in TetR HEK293 cells, using peaks in common in two replicates for each condition. b Motifs enriched in FOXM1 and GFP-FOXM1. Selected motifs identified in FOXM1 are also found in the GFP-FOXM1 peaks (full list in Additional file 1: Table S2). P values and percentages in brackets represent the statistical significance and the percentage of peaks for each motif, respectively. Motif sequences are sorted according to their proportional representation in FOXM1 peaks. c Examples of genomic regions showing three representative sites in common for endogenous FOXM1 and GFP-FOXM1 binding. d CEAS analysis comparing genomic distribution of endogenous FOXM1 binding events to GFP-FOXM1 in HEK293 cells