Mapping of binding sites in the PCE region of the AFP promoter. (A) Oligonucleotides L, S2, S1, R, M2, M1 and PCE, spanning the AFP promoter region from −177 to −150, are aligned with NK2 family- specific and FtzF1 family-specific oligonucleotides and with derived consensus binding sites for these families. The NK2 consensus is shown in both its direct and inverted forms. (B) Gel shifts were carried out using a recombinant peptide consisting of the Nkx2.8 homeobox and conserved region (aa 82 to 181), H441 cell, or αT3 cell extracts. One hundred and fifty nanograms of recombinant peptide or ∼6 μg of cell extract protein was utilized. The arrows indicate the positions of TTF1- and SF1-specific bands, and the asterisk shows the position of an upper band caused by binding of a second Nkx2.8 peptide. A strong αT3 cell band shift at a lower position was also apparent, but it was not further characterized since it did not correspond to an activity present in hepatocytic cell lines.

Transfection experiments. (A) Comparison of an intact AFP promoter and an AFP promoter with a partial deletion. CAT gene reporter plasmids consisted of the AFP enhancers combined with the intact (pAFP-246) AFP promoter or the AFP promoter (pAFP-166) with a partial deletion. These were expressed alone or cotransfected with CMV promoter plasmids expressing SF1, FTF, DAX1, Nkx2.8, or TTF1, all at an expression-to-reporter ratio of 0.33 to 1.0. Plots show the mean and standard deviation of four determinations. (B) DAX1 inhibition of FtzF1 factors. Reporter plasmid pPCE4 was transfected into AML12 cells alone or in combination with expression plasmids at a ratio of 0.33 to 1.0. Plots show the mean and standard deviation of four determinations. The reporter had detectable basal expression, probably due to a low level of endogenous FTF expression. (C) Effects of factor concentration on AFP promoter expression. pAFP-246 was cotransfected with various amounts of expression plasmid. The plots show the average of two determinations normalized to the expression of the reporter plasmid alone (1.0). (D) Detection of transcriptional activation. Reporter plasmids pS2-CAT (with FTF and SF1) and pC5-E1bCAT (with Nkx2.8, Nkx2.5, and TTF1) were cotransfected with various amounts of expression plasmid. The plots show the average of two determinations normalized to the expression of the reporter plasmid alone (1.0).

Antisense effect on secreted AFP. HuH7 cells plated in 3-cm microwells were transfected with oligonucleotides and were incubated overnight. The medium (1 ml) was then changed and collected after 24 h. Human albumin and AFP were quantified using commercial ELISA kits. The plots show the mean and standard deviation of six determinations from three separate platings normalized to the expression of plasmids transfected alone (100%). AFP AS and S results were compared at each concentration using Student's t test.

Binding of native cell extracts. (A) Demonstration of Nkx2.8 in nuclear extracts. Nuclear extracts, gel shift, and supershift assays were carried out as described in Materials and Methods, utilizing the NK2-specific binding oligonucleotide AACACTTGACT. Supershifts utilized an antibody to the Nkx2.8 C-terminal. (B) Binding by 8994 cell nuclear extract. Binding of two labeled oligonucleotides (Oligo) is shown with a series of specific competitions (Comp). The oligonucleotides were described in the Fig. 1 legend. (C) Binding by Hep3B cell nuclear extract. Gel shifts were obtained with nuclear extract (∼10 μg of protein) prepared as described in Materials and Methods. For the PCE gel shift pattern, a band with the approximate mobility of Nkx2.8 and FTF binding is marked with an arrow, and two regions that show a level of self-competition indicating specific binding are marked with filled circles.

Antisense oligonucleotides and cotransfection with reporter plasmids. (A) Oligonucleotides. The positions of phosphorothioate antisense (AS) and a control sense (S) strand oligonucleotide are aligned with a map of the Nkx2.8 mRNA. (B) Dual transfection. Plasmid DNA (pAFP6000CAT or pAFPE-Alb123CAT) was transfected into HuH7 cells using Lipofectamine PLUS; 3 h later, the cells were washed and oligonucleotides were transfected using Oligofectamine. Cells were collected for CAT assay after an additional 40 h. The plots show the mean and standard deviation of six determinations from three separate platings, normalized to the expression of plasmids transfected alone (100%). The pAFP6000CAT AS and S control oligonucleotide results were compared using Student's t test. (C) Gel shift analysis. Nuclear extract was prepared from cells treated with either the AS 757 or the control S 1033 oligonucleotides, and comparative gel shifts were carried out as done for Fig. 2B and C. To discriminate the specific bands, alternate lanes show competition at ×50 magnification with the same oligonucleotide.

Chromatin immunoprecipitation. Sheared formalin-fixed chromatin was prepared from HuH7 cells and precipitated with antibodies directed against the homeodomain and C-terminal regions of Nkx2.8, the NF-Y A subunit, HNF1α, or HNF1β. Each comparison represents a single precipitation followed by PCR detection of the albumin (Alb) (209 bp) and AFP (240 bp) promoters. A marker lane at left shows bands of 476, 341, 258, and 141 bp.