Figure 4.
NFATc2 binds to the p15INK4b promoter to mediate its silencing. (A) ChIP analysis using a specific NFATc2 antibody was performed in PaTu8988t cells after 1 hour of treatment with 10% fetal calf serum (FCS). In vivo binding of NFATc2 to the proximal p15INK4b promoter was determined by quantitative real-time PCR using primers specific for the wild-type (wt) p15INK4b promoter region harboring the NFATc2 binding site. (B) PaTu8988t cells were transfected with the wt p15INK4b promoter construct along with increasing amounts of wt NFATc2. Firefly luciferase activities were normalized to Renilla luciferase activity and expressed as fold induction relative to the activity of untreated p15INK4b promoter. (C) Schematic representation of the −751/+75 human full-length p15INK4b promoter construct (wt p15INK4b) and the indicated deletion constructs (p15INK4b del-I-III). The GGAAA NFAT consensus sequence is depicted in the wt p15INK4b promoter construct and also displays the NFAT binding site mutation shown as p15INK4b NFAT-mut. (D) PaTu8988t cells were transfected with the wt p15INK4b or the indicated deletion constructs (p15INK4b del I-III), and responsiveness was measured in the presence or absence of NFATc2. Reporter gene activities are expressed as fold induction related to the control transfected p15INK4b promoter constructs. (E) DNA pull-down experiment to characterize the binding of NFATc2 on the proximal p15INK4b promoter using nuclear extracts from PaTu8988t cells along with either wt p15INK4b or p15INK4b NFAT-mut oligonucleotide sequences. NFATc2 binding was analyzed by immunoblotting. (F) The luciferase activities of the wt p15INK4b and p15INK4b NFAT-mut promoter constructs in PaTu8988t cells were measured in the presence or absence of NFATc2. Reporter gene activities are expressed as fold activation of the p15INK4b promoter constructs relative to the induction without NFATc2.