Figure 5.
c-Myc Interacts with HNF4α1 in Vivo and in Vitro and Is Associated with Sp1 and HNF4α Binding Sites in the p21 Promoter
A, Co-IP of endogenous c-Myc and HNF4α1 using nuclear extracts from HepG2 cells (top panel) and of ectopically expressed c-Myc and HNF4α1 using whole-cell extracts from HEK293 cells transfected with Flag.c-Myc in the absence (c-Myc only) or presence of pMT7.HNF4α1 (c-Myc + H4) (bottom panel). The co-IP was performed using antibodies that recognize the amino terminus of c-Myc (top, lane 3), the Flag epitope (bottom, lanes 3 and 6), or control IgG. The IB was performed using the anti-HNF4α (α-445 or conjugated α-445-HRP) antibody. B, GST pull-down assays using in vitro-synthesized 35S-labeled c-Myc and immobilized GST, GST.HNF4α1 (FL, full-length; DBD; DBD/H, DBD plus hinge region; LBD; LBD/F, LBD plus F domain; and F, F domain alone) fusion proteins as indicated. The autoradiogram of the SDS gel after transfer to Immobilon membrane is shown as well as the Coomassie stain of the same blot showing relatively equal loading of the GST proteins. Bands corresponding to the GST fusion proteins are indicated by arrowheads. C, ChIP assays performed as in Fig. 3 using HCT116 wt cells not treated (Sp1), infected with Ad.HNF4α1 (HNF4α1), or transfected with Flag.c-Myc and harvested 24 h after doxycycline induction or transfection, as appropriate. Anti-Sp1 (Sp1), anti-HNF4α (H4, α-445), anti-Flag (Flag), and control antibodies (IgG) and the p21 primer sets described in Fig. 3A were used. D, ChIP assays performed in HepG2 cells 28 h after transfection with Flag.c-Myc, using the anti-Flag (Flag) and control IgG antibodies and the p21 primer sets described in Fig. 3A. C and D, Ratio of the signal from the specific antibodies to the IgG control is given.