In vitro DNase I footprint analysis of the hNT/N promoter. A, the coding and noncoding strands of an hNT/N gene fragment, from nucleotides −373 to +26, were labeled with [³²P]CTP, incubated with nuclear extracts from BON cells, and digested with DNase I. Areas protected from digestion are bracketed with the corresponding nucleotide positions, which was determined by running a DNA sequencing ladder (G reaction) in parallel. The numbers at the top of the autoradiogram are the amounts of nuclear extract (in μg of protein) used in each reaction mixture. B, sequences of the hNT/N promoter fragment and DNase I-protected regions I and II (shown as white letters on black background).

Site-directed mutagenesis analysis of region I. A, schematic representation of site-directed mutagenesis of region I. B, the wild-type region I (WTI) hNT/N promoter fragment (−60 to −36) and mutant fragments (M1–M4) were radiolabeled and incubated with BON cell nuclear extracts (10 μg). and EMSA was performed as described under “Experimental Procedures.” C, competitive binding analysis by EMSA with BON nuclear extracts (10 μg) and unlabeled WTI and mutant M1–M4 probes (at 200-fold molar excess) as competitors. D, the −175 to +26 region of the hNT/N promoter and its mutations, M1–M4, were cloned into the luciferase reporter plasmid, pXP1, and transiently transfected into BON cells. Luciferase activities are expressed as a percentage of the wild type (−175 to +26) fusion plasmid and are the mean ± S.D. of four separate transfections after normalization to β-galactosidase expression.

NR2F2 represses hNT/N promoter activity. A, BON cells were co-transfected with hNT/N promoter plasmids (−373, −122, and −71) and expression plasmids for NR2F2 (pMT-2ARP-1) or with empty vector (pMT-2-null) and a β-galactosidase plasmid (internal control). Luciferase activities are expressed as a percentage of the hNT/N plasmids (−373, −122, −71) without the NR2F2 construct (*, p > 0.05 hNT/N plasmid alone). B, BON cells were co-transfected with hNT/N promoter plasmids (−373, −122, and −71) and NR2F2 siRNA or nontargeting control siRNA (Dharmacon) and with pRL-RK (Promega, internal control) using the DharmaFECT Duo transfection reagent (Dharmacon). Luciferase activities are expressed as a percentage of hNT/N promoter activity in the presence of control siRNA and are the mean ± S.D. of three separate transfections after normalization to Renilla luciferase (pRL-TK) expression. C, stable NR2F2 knockdown BON cell lines were established as shown by the Western blot. D, real-time PCR was performed to quantify NT/N mRNA levels in BON cells with stable NR2F2 knockdown (*, p > 0.05 versus NTC-sh). E, an NT EIA assay was performed to measure basal (left panel) and FSK-stimulated (right panel) NT secretion in BON cells with NR2F2 knockdown (*, p > 0.05 versus NTC-sh; *†, p > 0.05 versus NTC-sh plus FSK).

Identification of the functional domains for NR2F2. A, schematic representation of the ARP-1 (NR2F2) deletion mutants used. B, expression plasmids for ARP-1 (NR2F2) and its mutants (Δ1, Δ6, and Δ7) or members of the nuclear receptor superfamily (RARα/β and RXRα/β) and the empty vector, pMT-2-null, were transfected into COS-1 cells. After 48 h, crude protein extracts (20 μg) from transfected or untransfected COS-1 cells were incubated with [³²P]ATP-labeled WTII probe for EMSA analysis. C, DNase I footprinting of the NR2F2 site. The coding strand of the hNT/N promoter (−373 to +26) was labeled, incubated with crude protein from COS-1 cells transfected with or without ARP-1 (NR2F2) and its mutants (Δ1, Δ6, and Δ7), and then digested with DNase I. Areas protected from digestion are bracketed with the corresponding nucleotide positions, which were determined by running a DNA sequencing ladder (G reaction) in parallel. The numbers at the top of the autoradiogram are the amounts of nuclear extract (in μg of protein) used in each reaction mixture. A 100-fold molar excess of the oligonucleotide corresponding to the hNT/N promoter fragment −106 to −77 was used as a competitor (lane 7). D, BON cells were co-transfected with the −122 hNT/N promoter plasmid and expression plasmids for NR2F2 (pMT-2-ARP-1) or its deletion mutants (Δ1, Δ6, and Δ7) with the empty vector, pMT-2, and the β-galactosidase plasmid (internal control). Luciferase activities are expressed as the percentage of the hNT-122 plasmid without NR2F2 and are the mean ± S.D. of four separate transfections after normalization to β-galactosidase expression.

5′-deletion and linker scanner (replacement) mutation analysis of hNT/N promoter activity. A, the region spanning 373 bp of the hNT/N promoter and the first 26 bp of exon 1 was progressively deleted from its 5′-end, fused to the luciferase-reporter vector pXP1, and transiently transfected into BON cells. Luciferase activities are expressed as the percentage of the −373 fusion construct and are the mean ± S.D. of four separate transfections after normalization to β-galactosidase expression. B, relative luciferase expression generated by wild type (−175 to +26) or the linker scanner (replacement) mutants of the hNT/N promoter after transfection into BON cells. Luciferase activities are expressed as a percentage of that of the wild type (−175 to +26) fusion plasmid and are the mean ± S.D. of four separate transfections after normalization to β-galactosidase expression.

Identification of the AP-1 and CREB/ATF proteins that bind to region I (−54 to −38) of the hNT/N promoter. Nuclear extracts (10 μg/lane) from BON cells were preincubated with antisera (2 μl) to various AP-1 and CREB/ATF proteins prior to the addition of labeled WT region I probe. Supershifted complexes were noted for c-Jun (lane 4), JunD (lane 5), ATF-1 (lane 11), ATF-2 (lane 12), and CREB (lane 13).

Identification of the NR2F2 protein that binds to region II (−100 to −76) of the hNT/N promoter. A, competitive binding analysis by EMSA with BON cell nuclear extracts (10 μg) and wild-type region II (WTII) as the probe. Unlabeled oligonucleotides containing consensus sites for GAGA, GATA, and HNF-1 were used as competitors at 30- and 300-fold molar excess. In addition, unlabeled WTII and oligonucleotide C3P, which contains a site that binds to both NR2F2 and HNF-4, were also used as competitors. B, nuclear extracts (10 μg/lane) from BON cells were preincubated with antisera (2 μl) to the NR2F2 and HNF-4 proteins prior to the addition of radiolabeled WTII. Supershifted complexes were noted for NR2F2 (lane 6). C, overexpressing plasmids of NR2F2 or HNF-4 and the control vector, pMT2, were transfected into COS-1 cells. Protein extracts from BON cells and transfected COS-1 cells were incubated with labeled WTII for EMSA analysis. Unlabeled WTII and oligonucleotide C3P were used as competitors at 200-fold molar excess. D, protein extracts from COS-1 cells transfected with NR2F2 or HNF-4 overexpression plasmids were preincubated with antisera to NR2F2, NR2F1, or HNF-4 prior to the addition of [³²P]ATP-labeled oligonucleotide C3P. Supershifted complexes were noted for NR2F2 (lane 4), HNF-4 (lane 7), and NR2F1 (lane 5). E, BON cells were subjected to ChIP assay; soluble chromatin was prepared from BON cells transfected with NR2F2 and immunoprecipitated (IP) with NR2F2 antibody (lane 2) or IgG (lane 3). Total (Input, lane 1) and immunoprecipitated DNAs were then PCR-amplified using primer pairs covering the NR2F2 binding site within the hNT/N promoter or GAPDH gene (control). F, Western blotting analysis was performed to detect NR2F2 expression level in BON cells compared with NCI-727 and QGP-1 cells. G, a ChIP assay was performed as described in E by precipitating endogenous NR2F2, CREB1, CREB2, ATF1, ATF2, JunD, and c-Jun (top panel). Densitometric analysis composes the -fold change of PCR products to IgG from two independent PCR analyses.

Site-directed mutagenesis analysis of region II. A, schematic representation of site-directed mutagenesis of region II. B, the wild type (WTII) hNT/N promoter fragment (−106 to −77) and its mutants (M5–M8) were radiolabeled and incubated with BON cell nuclear extracts (10 μg), and was performed as described under “Experimental Procedures.” C, competitive binding analysis by EMSA with BON nuclear extracts (10 μg) and unlabeled WTII and mutant probes M5–M8 (at 200-fold molar excesses) as competitors. D, the −175 to +26 region of the hNT/N promoter and its mutants (M5–M8) were cloned into the luciferase reporter plasmid, pXP1, and transiently transfected into BON cells. Luciferase activities are expressed as a percentage of the wild type (−175 to +26) fusion plasmid and are the mean ± S.D. of four separate transfections after normalization to β-galactosidase expression.