Figure 6: DDX3 interacts with CBP/p300 and induces the acetylation status of HNF4.

(a) DDX3 interacts with CBP and p300 in vivo. HuH7 cells were transfected with HA-DDX3 expressing plasmid or vector control. After 48 hr, the nuclear fractions (200 μg) were collected and incubated with antibodies against CBP or p300, and then the mixtures were bound to protein G sepharose beads. The precipitates were subjected to immunoblotting with anti-HA antibody. The IgG-conjugated protein G sepharose beads (mouse, lane 3 and lane 8) incubated with HA-DDX3 expressed nuclear extracts were used as negative control. Input; 10% (20 μg) of the nuclear extract. (b) Ectopic expression of DDX3 induces the acetylation status of HA-HNF4. HuH7 cells were transfected with plasmid expressing HA-HNF4 alone or together with HA-DDX3 expression construct as indicated. The whole cell extracts (1 mg) prepared from the transfected cells were subjected to immunoprecipitation of HA-HNF4 with anti-HA agarose beads. After extensive wash, the total amounts and acetylated forms of immunoprecipitated HA-HNF4 were detected by Western blot analysis with anti-HA and anti-acetylated lysine (Cell Signaling Technology) antibodies, respectively. (c) In situ proximity ligation assay (PLA) indicates that ectopic expression of DDX3 induces endogenous HNF4 acetylation. HuH7 cells transfected with either GFP or GFP-DDX3 expressing plasmids for 48 hr were fixed and subjected to PLA with anti-HNF4 (Abcam) and anti-acetylated lysine antibodies. Anti-NS5A antibody (Austral Biologicals) served as control. Nuclei were stained with DAPI (blue). The numbers of PLA signals per cell were quantified using MetaMorph software (Molecular Devices) and are shown as means ± S.D. relative to that of GFP-transfected cells. n = 50. ***p < 0.001.