Involvement of HNF-4α and FoxA2 in apoAI gene transcription. A, apoAI RNA expression in 293T (lane 1), HeLa (lane 2), and HepG2 (lane 3) was analyzed by reverse transcription-PCR. β-Actin RNA is shown for normalization (lanes 4–6). B, ChIP analyses to assess occupancy of the apoAI promoter-proximal enhancer by HNF-4α and FoxA2. Cells (293T, HeLa, or HepG2) were processed for a standard ChIP assay, and immunoprecipitates from the indicated antibodies were subjected to real time PCR using amplicon primers that specifically amplify either the apoAI promoter or a distal (3′) region as indicated. For each set, the data are plotted as fold-enrichment of the specific signal relative to the corresponding IgG control. C, synergistic activation of a luciferase reporter by HNF-4α and FoxA2. For the transient transfection assay using a luciferase reporter containing cognate sites for HNF-4α and FoxA2 in close juxtaposition, 293T cells were transfected with 100 ng of a FoxA2-expressing plasmid or 50 ng of an HNF-4α-expressing plasmid or a combination of the two, as indicated.

Physical interactions between FoxA2 and DNA-PK. A, for the GST pulldown assay, GST-FoxA2 (lane 3; fl, full-length) or control GST (lane 2) was immobilized on glutathione-Sepharose beads, incubated with HeLa nuclear extract, washed, and analyzed for retention of the indicated polypeptides by Western blotting. Input HeLa extract (2%) was also loaded on the gel. B, GST pulldown assay was done as in A except that phosphocellulose P11 0.5 m fraction was used as the input (lane 1). Eluates from control GST (lane 2) or GST-FoxA2(fl) (lane 3) were probed with anti-DNA-PKcs antibodies. C, Coomassie Blue staining of the GST fusion proteins used in the pulldown assay shown in D (control GST, lane 1; FoxA2-N, lane 2; FoxA2-WH, lane 3; and FoxA2-C, lane 4). The proteins were expressed in E. coli, purified over glutathione-Sepharose beads, and resolved by SDS-PAGE prior to staining. Note that because the FoxA2-C derivative tended to degrade, normalization was based on the full-length products (arrow) rather than total protein amounts. For exact coordinates of the constructs, see also the schematic of Fig. 5B. D, GST pulldown assay was performed as described for A, except that truncated FoxA2 derivatives were bound to the beads, as indicated. GST alone (lane 2) served as a control, and 2% of the HeLa extract input was also loaded on the gel.

Phosphorylation of FoxA2 by DNA-PK. A, full-length bacterially expressed FoxA2 (lane 1, FoxA2 WT) and the corresponding mutant (see text for details) in the mapped DNA-PK phosphorylation site (lane 2, FoxA2(T280A,S283A)) were incubated with the catalytic subunit of DNA-PK in the presence of [γ-³²P]ATP. The reaction products were visualized by autoradiography (autorad, bottom panel). Top panel shows a Western blot (WB) of the two purified proteins. Arrows indicate full-length FoxA2 in the preparations, which tend to degrade. B, schematic diagram (adapted from Ref. 18) showing the domain organization of FoxA2, including the central winged helix DNA binding domain and the N- and C-terminally located activation domains (AD). The amino acid coordinates of various derivatives used in this study are also indicated. Amino acid sequences (human and rat) of corresponding linker regions are just adjacent to the winged helix domains that appear to be targeted by DNA-PK (C and D). The serine residue identified as the DNA-PK target site within FoxA2 lies within a conserved DNA-PK signal (TQ/SQ) and is circled. Note that in our FoxA2(T280A,S283A) construct, the T280A change (in addition to the S283A) also eliminates any disparities that may be attributable to differences between the rat and human sequences. C, identification of the FoxA2 domain that is phosphorylated by DNA-PK phosphorylation. FoxA2 derivatives (FoxA2-N, lane 1; FoxA2-WH, lane 2; FoxA2-C, lane 3) were subjected to DNA-PK- mediated phosphorylation as in A. Reaction products were resolved by SDS-PAGE and visualized by both Coomassie Blue staining (to ensure that equivalent amounts of the polypeptides were loaded; upper panel) and by autoradiography (lower panel). Bands corresponding to the test proteins are identified (N, FoxA2-N; WH, FoxA2-WH; C, FoxA2-C) for the upper panel. Arrows mark the phosphorylated FoxA2-C derivative for the lower panel. D, mapping of the serine residue in the FoxA2-C domain that is phosphorylated by DNA-PK. The indicated derivatives of the FoxA2-C domain were analyzed for their ability to be phosphorylated by DNA-PK as described for C. These derivatives include the following: FoxA2-CΔ (lane 2), which contains an 11-residue N-terminal deletion that removes the putative DNA-PK sites (TQ/SQ); FoxA2-C(S283A,T280A; lane 3), a double point mutant in which both the serine and threonine are mutated; and FoxA2-C(S283A; lane 4), a point mutant in which the serine is mutated to alanine.

Effect of ablation of the DNA-PK phosphorylation site on transcriptional activation by FoxA2. A, B3XAI.LUC reporter plasmid (100 ng) was transfected into HepG2 cells together with the indicated amounts (in nanograms) of vectors expressing FoxA2 WT or FoxA2(T280A,S283A). Luciferase activity was normalized as described for Fig. 6, and the data are expressed as fold-activation relative to control transfections with no ectopic FoxA2. B, luciferase reporter containing binding sites for both HNF-4α and FoxA2 was transfected into HepG2 cells together with indicated amounts (in nanograms) of vectors expressing HNF-4α and either FoxA2 WT or FoxA2(T280A,S283A). The results were analyzed as described for A. C, Western blot analysis of transfected FoxA2 proteins. HepG2 cells were transfected with the control vector alone (lane 1) or vectors expressing FLAG-tagged FoxA2 WT (lane 2) or FLAG-tagged FoxA2(T280A,S283A) (lane 3). After 48 h whole cell lysates from the cells were analyzed by Western blotting using an anti-FLAG antibody.

DNA binding analysis of FoxA2 to its target site. A, EMSA showing binding of recombinant FoxA2 to a site B probe (lane 1) and its competition by increasing amount of unlabeled site B oligonucleotide (lanes 2–4) but not equivalent amount of site A oligonucleotide (lanes 5–7). The molar ratios of cold competitor over labeled probe were as follows: lanes 2 and 5, 2.5; lanes 3 and 6, 10; lanes 4 and 7, 50. B, further validation of the FoxA2 EMSA using an anti-FoxA2 antibody (lane 2) to supershift the specific complex (identified by the arrow). As control, total goat IgG was used (lane 1). C, increasing amounts of FoxA2 WT (lane 1, 1.6 ng; lane 2, 4 ng; lane 3, 8 ng) or FoxA2(T280A,S283A) (lane 4, 2.8 ng; lane 5, 7 ng; lane 6, 14 ng) were incubated with the site B probe and analyzed by EMSA. D, using the indicated bodies, ChIP assays were performed on extracts of 293T cells that had been cross-linked with formaldehyde 48 h after transfection with the B3XAI.LUC reporter and either the empty vector (pVP5; top panel), the vector expressing wild-type FoxA2 (middle panel), or the vector expressing the mutant FoxA2 protein (lower panel). Immunoprecipitate cross-links were reversed, and the promoter region on the reporter template was amplified by PCR.

Copurification and specific association of FoxA2 and DNA-PK. A, SDS-PAGE analysis (silver-stained gel is shown) of a FLAG peptide-eluted preparation obtained after M2-agarose affinity purification of nuclear extract derived from a cell line that stably expresses f:FoxA2 (lane 2). A corresponding eluate from a control cell extract that does not express f:FoxA2 was also run on the gel (lane 1). Molecular mass markers (in kDa) are shown just left of the column. The f:FoxA2 polypeptide as well as the apparently specifically associated polypeptides with molecular masses estimated to be ∼100 kDa and more than 350 kDa are indicated. B, SDS-PAGE analysis (visualized by silver staining) of the preparation shown in A, lane 2, that was additionally resolved on a Superose 6 gel filtration column. Every other fraction in the main protein peak was loaded on the gel. Bands identified as f:FoxA2 and p > 350 are marked (see also D). C, specificity of association between FoxA2 and DNA-PK. The eluates shown in A (control, lane 3; f:FoxA2, lane 4) were analyzed by Western blot using antibodies against DNA-PKcs (upper panel) and FoxA2 (lower panel). Standard unfractionated nuclear extracts from HepG2 (lane 1) and HeLa (lane 2) were also included. Note that HepG2 cells express FoxA2, but at the sensitivity of the Western shown, the corresponding band is not visible in lane 1. D, Superose 6 fractions shown in B were analyzed by Western blot using antibodies against DNA-PKcs (upper panel) and FoxA2 (lower panel).

Intracellular interactions between FoxA2 and DNA-PK. A, coimmunoprecipitation assays utilized whole cell extracts (WCE) from 293T (lane 1) and HepG2 (lane 2) cells. The extracts were incubated with control antibodies (goat IgG, lanes 3 and 5) or with anti-FoxA2 (lanes 4 and 6) antibodies. After washing, the precipitates were analyzed by Western blotting with the indicated antibody (top panel, anti-DNA-PKcs; bottom panel, anti-FoxA2). Nonspecific bands in the immunoprecipitates (IP), which likely result from cross-reactivity to immunoglobin heavy chains, are marked with asterisks. Note that whereas immunoblots shown for lanes 3–6 are from the same experiment, those showing the inputs used for this experiment (lanes 1 and 2) are from a separate run. B, ChIP analysis to assess occupancy of the apoAI promoter-proximal enhancer by DNA-PK. HepG2 cells were processed for a standard ChIP assay and immunoprecipitated with either control IgG or an anti-Ku70 antibody. The processed samples were subjected to real time PCR using amplicon primers that specifically amplify either the apoAI promoter or a distal region as in Fig. 1B. Data are plotted as enrichment of the specific signal relative to the IgG control for each amplicon.

Effect of DNA-PK on transcriptional activation by FoxA2. A, B3XAI.LUC reporter plasmid (100 ng) was transfected into either SCH8 (DNA-PKcs^+/+, black bars) or SCSV3 cells (DNA-PKcs^−/−, gray bars) either alone or together with 250 ng of a FoxA2-expressing plasmid. For each cell type, luciferase activity was first normalized using the corresponding Renilla reading. Taking the normalized activity in the absence of ectopically expressed FoxA2 as basal (hence, 1.0), the data were expressed as fold-activation in the context of the indicated cell line. Student's t test was used to evaluate the data (p = 0.09; n = 4). B, to authenticate the phenotypes of the two cell lines, whole cell extracts from SCH8 (lane 1, DNA-PKcs^+/+) or SCSV3 (lane 2, DNA-PKcs^−/−) were analyzed by Western blotting using either antibodies against DNA-PKcs (upper panel) or TAF100 (lower panel). C, SCSV3 (DNA-PKcs^−/−) cells contain a kinase that also can phosphorylate the FoxA2-C derivative. For the in vitro kinase assay purified FoxA2-C (lanes 2 and 4) was incubated with extracts from SCH8 (DNA-PKcs^+/+, lanes 1 and 2) or from SCSV3 (DNA-PKcs^−/−; lanes 3 and 4) cells in the presence of [γ-³²P]ATP. Following the reaction, the samples were purified over nickel-nitrilotriacetic acid-agarose beads, concentrated by trichloroacetic acid precipitation, and resolved by SDS-PAGE. Labeled proteins (arrow) were visualized by autoradiography.

Ablation of the DNA-PK phosphorylation site on FoxA2 does not alter its subcellular localization. Representative images of immunostained 293T cells transfected with either vector control, FoxA2 WT, or FoxA2(T280A,S283A) are shown. FoxA2 protein (green) is localized to the nuclei, which were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) (blue). There was no detectable staining of FoxA2 in vector control cells.