Table 1.
Wild-type and mutated sequence of constructs used in mutational analysis.
Table 2.
Sequences of double-stranded oligonucleotides used in EMSA.
Fig 1.
HepG2-specific regulation of the hDIO1 promoter by T0901317 (TO).
A series of 5´-deletion constructs of the hDIO1 promoter were transiently transfected into HepG2 and TSA201 cells along with expression vectors for human LXRα and human RXRα (LXRα/RXRα) with and without 10−7 M TO. Promoter activity was normalized against Renilla luciferase activity, and the normalized value is expressed relative to that of promoterless pGL 4.10 in the absence of TO. Results are expressed as means ± SEM. *, P < 0.05; **, P < 0.01. A. Basal luciferase activity of each construct. Statistical analysis was performed on pairwise comparisons of constructs, and significant pairs are presented. B. Luciferase activities of each construct with and without 10−7 M TO. TO induction indicates ratio of promoter activity with TO to the activity without TO. Statistical analysis was performed to compare TO induction of each construct with that of promoterless pGL 4.10, and significant differences are presented.
Fig 2.
Analysis of relative hDIO1 mRNA levels in HepG2 cells.
hDIO1 mRNA levels were normalized against the corresponding levels of cyclophilin A mRNA, and the value in HepG2/vehicle cells was defined as 1. Values are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; N.S., not significant. A. HepG2 cells were treated at different concentrations of TO for 24 h. B. HepG2 cells were treated with vehicle, 10−6 M TO, or 10−6 M TO and 10−6 M GSK2033 (GSK) for 24 h. C. HepG2 cells were exposed to cycloheximide for 30 min before being treated with TO (10−7 M) for 24 h.
Fig 3.
Mutational analysis of activation of the hDIO1 promoter by T0901317 (TO).
A. The nucleotide sequence of the 5´-flanking region of the hDIO1 promoter used in the analysis, along with the positional relationship among mutated oligonucleotides, thyroid hormone response element (TRE), and the putative activator protein 1 (AP-1) site. † and black horizontal bars represent the site-specific mutation. B and C. A series of mutated hDIO1 promoter constructs was transiently transfected into HepG2 cells along with expression vectors for LXRα/RXRα, with or without 10−7 M TO. Schematic diagram to the left of the figure representing mutant (No. 1–5) and wild-type (No. 6) hDIO1 promoters, which were introduced upstream of the luciferase gene. No. 7 represents the promoterless pGL 4.10 construct. Promoter activity was normalized against Renilla luciferase activity, and is expressed relative to that of promoterless pGL 4.10 in the absence of TO. Values are expressed as means ± SEM. *, P < 0.05; **, P < 0.01; N.S., not significant. B. Basal luciferase activity of each construct. Statistical analysis was performed on comparisons between all constructs. Because most pairs exhibited significance, only non-significant pairs are presented. C. Luciferase activities of each construct with and without 10−7 M TO. TO induction indicates ratios of promoter activity with TO to the activity without TO. Statistical analysis was performed on comparisons of TO induction of each construct with that of promoterless pGL 4.10, and significant differences are presented.
Fig 4.
Specific binding of transcription factors to the region between nucleotides −131 and −114 of the hDIO1 promoter.
A. EMSA with oligonucleotide containing the wild-type sequence of the region between nucleotides −141 and −112 of the hDIO1 promoter (Wt1) and nuclear extracts from un-treated HepG2 cells and TSA201 cells. In lane 1, as a control, only the biotin-labeled Wt1 oligonucleotide was present. Biotin-labeled Wt1 oligonucleotide was incubated with nuclear extracts from HepG2 or TSA201 cells without competitors in lanes 2 and 4, respectively, and with 25-fold molar excesses of unlabeled Wt1 oligonucleotides in lanes 3 and 5, respectively. The specific DNA/protein complexes formed are indicated by arrows. B. EMSA with mutant oligonucleotides and nuclear extracts from HepG2 cells. Biotin-labeled Wt1 oligonucleotide was incubated with nuclear extracts from HepG2 cells without competitors in lane 2, and with 25-fold molar excesses of unlabeled Wt1, −124mut1, and −126/−125mut1 oligonucleotides in lanes 3, 4, and 5, respectively. The specific DNA/protein complexes formed are indicated by arrows.
Fig 5.
Binding of LXRα on the hDIO1 promoter.
A. EMSA with oligonucleotide containing the wild-type sequence of the region between nucleotides −141 and −112 of the hDIO1 promoter (Wt1) with nuclear extracts from vehicle-treated (V), T0901317 (TO)-treated (T), or TO-treated and LXRα/RXRα-overexpressing (T+LXRα/RXRα) HepG2 cells; also shown is a supershift assay with an antibody against LXRα. Specific DNA/protein complexes are indicated by arrows. In lane 1, as a control, only biotin-labeled Wt1 oligonucleotide was present. In lanes 2, 3, and 4, biotin-labeled Wt1 oligonucleotide was incubated with nuclear extracts from V, T, and T+LXRα/RXRα HepG2 cells without competitor, respectively. In lane 5, biotin-labeled Wt1 oligonucleotide was incubated with nuclear extracts from T+LXRα/RXRα HepG2 cells with excess unlabeled Wt1 oligonucleotides as competitor. The results of the supershift assay are shown with normal mouse IgG as a control in lane 6 and with an antibody against LXRα in lane 7. B. EMSA with Wt1 oligonucleotides with nuclear extracts from T+LXRα/RXRα HepG2 cells and a supershift assay with antibodies against TRβ and RXRα. Specific DNA/protein complexes are indicated by arrows. In lane 1, as a control, only biotin-labeled Wt1 oligonucleotide was present. Biotin-labeled Wt1 oligonucleotide was incubated with the nuclear extracts in lane 2 and with the nuclear extracts and excess unlabeled Wt1 oligonucleotides as competitor in lane 3. The results of the supershift assay are shown with normal mouse IgG as a control in lane 4, with an antibody against TRβ in lane 5, and with an antibody against RXRα in lane 6. C. EMSA was performed with oligonucleotides containing the wild-type sequence of the region between nucleotides −131 and −104 of the hDIO1 promoter (Wt2) with nuclear extracts from T+LXRα/RXRα HepG2 cells; also shown is a supershift assay with antibodies against LXRα, TRβ, and RXRα. A specific DNA/protein complex is indicated by an arrow. In lane 1, as a control, only biotin-labeled Wt2 was present. Biotin-labeled Wt2 was incubated with nuclear extracts in lane 2, with nuclear extracts and excess unlabeled Wt2 oligonucleotides as competitor in lane 3, and with excess unlabeled −126/−125 mut2 as competitor in lane 4. The results of the supershift assay are shown with normal mouse IgG as a control in lane 5, with an antibody against LXRα in lane 6, with an antibody against TRβ in lane 7, and with an antibody against RXRα in lane 8.
Fig 6.
Interaction of LXRα/RXRα and TRβ on the activity of the hDIO1 promoter.
HepG2 cells were transfected with or without expression vectors for LXRα, RXRα, or LXRα/RXRα, with or without TRβ or siRNA specific for THRB. We performed luciferase assay using a −131/−4 hDIO1-Luc construct. All upper figures show only basal luciferase activities, whereas lower figures show both basal activities and activities with or without 10−7 M TO. TO induction indicates ratios of promoter activity with TO to the activity without TO. Promoter activity was normalized against Renilla luciferase activity, and the normalized value is expressed relative to that of promoterless pGL 4.10 in the absence of TO. Results are expressed as means ± SEM. Statistical analysis was performed on comparisons among all conditions; the results of basal activity are shown in the upper figure, and those of TO induction are shown in the lower figure; *, P < 0.05; **, P < 0.01. A. Comparison with and without overexpression of LXRα or TRβ. B. Comparison with and without overexpression of RXRα or TRβ. C. Comparison with and without overexpression of LXRα/RXRα or TRβ. D. Comparison with and without overexpression of LXRα/RXRα and TRβ knockdown using siRNA. si, siRNA specific for THRB; N/C, negative control siRNA.