Figure 6.
HP1γ stabilizes the repression complex to silence p15INK4b expression. (A) Immunofluorescence staining was performed on transfection of PaTu8988t cells with HA-NFATc2 and GFP-HP1γ constructs and 1 hour of stimulation with 10% fetal calf serum (FCS). Arrows indicate the colocalization of HA-NFATc2 (red signal) and GFP-HP1γ (green signal) in euchromatic foci (weak 4,6-diamidino-2-phenylindole [DAPI] staining within the nucleus) of the nucleus. Nuclei were visualized by DAPI (blue signals) staining. (B) Coimmunoprecipitation to show the Suv39H1 dependency for NFATc2 and HP1γ physical interaction in PaTu8988t cells. (C) PaTu8988t cells were subjected to ChIP analysis after knockdown of Suv39H1 to confirm the requirement of Suv39H1 for HP1γ binding to the wild-type (wt) p15INK4b promoter. (D) DNA pull-down experiment showing the destabilization of NFATc2 and Suv39H1 binding to the wt p15INK4b promoter after knockdown of HP1γ. Successful knockdown is shown by Western blot analysis (lower panel). (E) ChIP analysis was performed to determine HP1γ (left panel) or NFATc2, Suv39H1, and H3K9-me (right panel) binding to the wt p15INK4b promoter following silencing of HP1γ. Successful knockdown was confirmed by Western blotting. (F) Reporter gene assay to prove that the abolishment of HP1γ expression reverses p15INK4b silencing by NFATc2. Reporter gene activities were expressed as fold luciferase activity related to Renilla luciferase activity ±SD.