Figure 5.

TIP60 represses HPV18 E6 mRNA. (A) Increase in HPV18 E6 mRNA after depletion of TIP60. HeLa cells were transfected with indicated siRNA and RNA was prepared from cells harvested 72 hrs after transfection. Q-RT-PCR was performed and normalized to actin and relative levels of HPV18 E6 mRNA is shown. Mean ± S.D. of three experiments. (B) Over-expression of TIP60 in HeLa cells. Flag-TIP60 was over-expressed in HeLa cells after infecting with a retrovirus and selecting the cells on puromycin. (C) HPV18 E6 mRNA is repressed in TIP60 over-expressing cells. Relative change in HPV18 E6 mRNA shown in the cells expressing either vector alone or Flag-TIP60. Mean ± S.D. of two experiments, (*p≤0.05). Rest as in (A) and (B). (D) HPV18 promoter is repressed by TIP60. Control firefly luciferase plasmid (pGL3C) or plasmid expressing firefly luciferase driven by the HPV18 major early promoter (pGL3C-HPV) were transfected into mock or TIP60 over-expressing HeLa cells. The ratio of the firefly luciferase to co-transfected renilla luciferase is calculated and normalized values are shown. Mean ± S.D. of two experiments, (**p≤0.05). (E) TIP60 binds to HPV18 promoter. ChIP assay was performed in HeLa cells to examine binding of TIP60 on HPV18 promoter. Ratio of Q-PCR signal in TIP60 immunoprecipitate relative to pre-immune (negative control) immunoprecipitate is plotted: mean ± S.D. of three experiments. (F) TIP60 acetylates histone H4 on HPV18 promoter. Level of acetylated histone H4 determined by ChIP assay and comparison between the control and TIP60 depleted cells is shown. Rest as in (E). (G) Depletion of TIP60 decreases tri-methylation on lysine 9 of histone H3. Rest as in (E)