Fig 6.

FOXM1 is recruited indirectly via the MMB complex to CHR elements. (A and B) GST pulldown assay using the indicated GST-FOXM1 fusion proteins and total cell extracts from U2OS cells. Ethidium bromide (EtBr) was added to the GST pulldown reactions where indicated. Interacting LIN9 and B-MYB proteins were revealed by Western blotting (upper panels) and Coomassie or Ponceau S stained input GST fusion proteins are shown below. A total of 3% cell lysate input is shown (lane 1). Arrows represent bands corresponding to full-length GST fusion proteins. Asterisks indicate a cross-reacting GST fusion protein band. (C) Coimmunoprecipitation (IP) analysis from U2OS cells expressing the indicated Flag-tagged FOXM1 fusion proteins. Precipitated and coprecipitated proteins were detected by immunoblotting with the indicated antibodies (left). A total of 3% cell lysate input is shown (lanes 1 to 3). (D) FOXM1(Δ1-116) is localized to the nucleus. U2OS cells were transfected with plasmids encoding Flag-tagged version of full-length wild-type (WT) and a Δ1-116 version of FOXM1, and expression was detected by immunofluorescence with anti-Flag antibody. Nuclei are revealed by using DAPI (4′,6′-diamidino-2-phenylindole) staining. A merge of the two stains is shown on the right. (E) ChIP assays of transiently transfected Flag-tagged FOXM1 in HEK293T cells. Cells were transfected with empty vector or wild-type (WT) or mutant (Δ1-116/ΔN) versions of FOXM1 prior to ChIP for Flag-tagged FOXM1 on the indicated genes. Expression levels were revealed by Western analysis with anti-flag antibody (inset). The data are the averages of two independent experiments. (F) Model showing recruitment of FOXM1 to the CHR element through MMB complex binding. FOXM1 is linked to the cell cycle through activation by cell cycle-regulated kinases.