![Figure 6. HIF-1α/ARNT heterodimers bind directly to the microsatellite in vitro and in vivo. EMSA was performed with nuclear extracts from THP-1 cells either untreated or treated for 6 hours with (A) 1 μg/mL E. coli LPS and (B) 100 nM fMLP. Alleles 1 to 7 microsatellites or the mutant (GT)22 were used for binding assays in panel A; shifts for only alleles 1 to 6 are shown in panel B. In both cases, lane 1 contains no nuclear extract (ie, a3 oligonucleotide only [−] and lane 2 (Un) contains untreated control nuclear extract; arrow shows specific HIF-1 binding. (C) In vitro–translated HIF-1α (lane 3), ARNT (lane 4) or HIF-1α/ARNT heterodimers (lanes 5 and 7) were incubated with untreated (lanes 3-6 and 8) or hexamine CoCl2-treated allele 3 microsatellite (lane 7). HIF-1α/ARNT heterodimers bound (arrows) to wild-type but not to the mutant microsatellite (GT)22 (lane 8). Binding was competitively inhibited by excess cold microsatellite DNA (lane 6). Lane 1 shows oligonucleotide only; lane 2, negative control with untranslated reticulocyte lysate; NS, nonspecific binding component(s); F, free probe in panels A-C. (D) Chromatin immunoprecipitation of the SLC11A1 promoter from THP-1 cells treated with LPS/IFNγ or zymosan was performed using an anti–HIF-1α antibody; a nonspecific antibody (NS) was used as negative control, and 10% input chromatin was used as positive control for PCR. (E) Yeast 1-hybrid assay for HIF-1α/ARNT occupancy and transactivation of the SLC11A1 promoter. A total of 4 independent GAL4BD-HIF-1α/GAL4AD-ARNT yeast cotransformants were tested for Leu-Trp-Ura prototrophy and for qualitative β-galactosidase expression (blue coloration) by colony-lift filter assay, after exposing the prototrophs to the β-galactosidase substrate X-gal for 4 hours. (F) Quantitative β-galactosidase assay. Yeast cells with a chromosomally-integrated copy of the promoter were either untransformed or transformed with GAL4BD-HIF-1α, GAL4AD-ARNT, or both, and β-galactosidase expression was determined by chemiluminescence. SD/Leu-Trp-Ura medium was used as background control. Error bars denote SEM.](https://ash.silverchair-cdn.com/ash/content_public/journal/blood/110/8/10.1182_blood-2006-12-063289/4/zh80210708440006.jpeg?Expires=1722706433&Signature=hSlxtsq8seTYtD1-D1y79AvQVMyqmoP9uGDmlEpwHvrZ4UIyyzNWAgWJN8hHS9dZwMoA6esIyHiESJbGf8rgE65Lr8UFNVO7IgtJ0dWI9xdUbb5S~LIVGSw8q1h5XyMzHkkN4uKFrl9KDFmL-yW82YMUdHJZObkE3TRC5cGWG1ZvyzocEGJLE80MkYUuIUqNeeLj5A1zynQZCVGM7XD~5hoxaX~Jcg0akciO1JO-Gvy2oAfQy~c3k4TZRv5nLPazhfScJXIfbp9mu85xG0QIaixIYZtszgNvhcjCewNTAruv~a~T1sUJ6I5Qfr-L5QIoHxPkAf783D3Ozpch~jxhzg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
HIF-1α/ARNT heterodimers bind directly to the microsatellite in vitro and in vivo. EMSA was performed with nuclear extracts from THP-1 cells either untreated or treated for 6 hours with (A) 1 μg/mL E. coli LPS and (B) 100 nM fMLP. Alleles 1 to 7 microsatellites or the mutant (GT)22 were used for binding assays in panel A; shifts for only alleles 1 to 6 are shown in panel B. In both cases, lane 1 contains no nuclear extract (ie, a3 oligonucleotide only [−] and lane 2 (Un) contains untreated control nuclear extract; arrow shows specific HIF-1 binding. (C) In vitro–translated HIF-1α (lane 3), ARNT (lane 4) or HIF-1α/ARNT heterodimers (lanes 5 and 7) were incubated with untreated (lanes 3-6 and 8) or hexamine CoCl2-treated allele 3 microsatellite (lane 7). HIF-1α/ARNT heterodimers bound (arrows) to wild-type but not to the mutant microsatellite (GT)22 (lane 8). Binding was competitively inhibited by excess cold microsatellite DNA (lane 6). Lane 1 shows oligonucleotide only; lane 2, negative control with untranslated reticulocyte lysate; NS, nonspecific binding component(s); F, free probe in panels A-C. (D) Chromatin immunoprecipitation of the SLC11A1 promoter from THP-1 cells treated with LPS/IFNγ or zymosan was performed using an anti–HIF-1α antibody; a nonspecific antibody (NS) was used as negative control, and 10% input chromatin was used as positive control for PCR. (E) Yeast 1-hybrid assay for HIF-1α/ARNT occupancy and transactivation of the SLC11A1 promoter. A total of 4 independent GAL4BD-HIF-1α/GAL4AD-ARNT yeast cotransformants were tested for Leu-Trp-Ura prototrophy and for qualitative β-galactosidase expression (blue coloration) by colony-lift filter assay, after exposing the prototrophs to the β-galactosidase substrate X-gal for 4 hours. (F) Quantitative β-galactosidase assay. Yeast cells with a chromosomally-integrated copy of the promoter were either untransformed or transformed with GAL4BD-HIF-1α, GAL4AD-ARNT, or both, and β-galactosidase expression was determined by chemiluminescence. SD/Leu-Trp-Ura medium was used as background control. Error bars denote SEM.
