Fig. 1.
Fig. 1. The CCAAT box is the critical component of the −65/−52 IL-6RE of the JunB promoter. / (A) The IL-6 responsive element of the JunB promoter was localized between −65 and −52 and termed the −65/−52 IL-6RE.1This element contains 2 motifs, a CCAAT box and an IR repeat. (B) Sequences of wild type and mutant oligonucleotides that were used in EMSA experiments and in the minimal promoter constructs. Sequences of mutant oligos were derived from the JunBMut fragment. Only the coding strand is indicated. Underlined sequence indicates the −65/−52 IL-6RE. Bold sequences indicate the mutated sequences. Double line indicates the CCAAT box and the thick solid line shows the IR region. Indicated position of the mutated sequences is relative to the transcription start site. (C) Binding analysis by EMSA of mutant oligonucleotides. Cold competition assays using nuclear extract from IL-6–stimulated (15 minutes) M1 cells and JunBWT as probe, with excess unlabeled competitor oligos (100-fold) added 5 minutes prior to the addition of probe. Control lane contains no competitor oligos. (D) In vivo CAT induction by IL-6 when mutant oligonucleotides are cloned into promoter region of JunB minimal promoter expression vector. Mutant oligonucleotides were cloned upstream from the −31GD minimal JunB promoter and constructs were transiently transfected into M1 cells, either untreated or treated with IL-6 (100 ng/mL). CAT activity was ascertained, as previously described.1 The sample numbers in D correspond to the sample numbers in C, with regard to the oligos used. Lane 7 indicates transfection with pCAT-Basic vector. Values are averages of 3 independent experiments, and SDs are indicated. (E) Methyl interference assay on the −65/−52 IL-6 response element to identify protein binding sites. Noncoding and coding strands were 5′ end labeled and the probe was methylated. Binding reactions were done with 50 000 cpm methylated probe and 20 μg nuclear extracts from cells treated with IL-6 for the indicated times. Resulting protein-DNA complexes were resolved on 5% nondenaturing polyacrylamide gel. After autoradiography, free and bound probes were excised and eluted from the gel. Eluted DNA was digested with piperidine for 30 minutes at 90°C and resolved on 12% DNA sequencing PAGE. F, Free Probe. Two potential binding sites were detected (indicated by open boxes). Area I covers the GGCCAATCG sequence and area II the CACTTC sequence. Schematic diagram of the JunBWT probe that was used is indicated. *Indicates G residues that are protected from cleavage based on labeling of the noncoding strand, and open stars, of the coding strand.

The CCAAT box is the critical component of the −65/−52 IL-6RE of the JunB promoter.

(A) The IL-6 responsive element of the JunB promoter was localized between −65 and −52 and termed the −65/−52 IL-6RE.1This element contains 2 motifs, a CCAAT box and an IR repeat. (B) Sequences of wild type and mutant oligonucleotides that were used in EMSA experiments and in the minimal promoter constructs. Sequences of mutant oligos were derived from the JunBMut fragment. Only the coding strand is indicated. Underlined sequence indicates the −65/−52 IL-6RE. Bold sequences indicate the mutated sequences. Double line indicates the CCAAT box and the thick solid line shows the IR region. Indicated position of the mutated sequences is relative to the transcription start site. (C) Binding analysis by EMSA of mutant oligonucleotides. Cold competition assays using nuclear extract from IL-6–stimulated (15 minutes) M1 cells and JunBWT as probe, with excess unlabeled competitor oligos (100-fold) added 5 minutes prior to the addition of probe. Control lane contains no competitor oligos. (D) In vivo CAT induction by IL-6 when mutant oligonucleotides are cloned into promoter region of JunB minimal promoter expression vector. Mutant oligonucleotides were cloned upstream from the −31GD minimal JunB promoter and constructs were transiently transfected into M1 cells, either untreated or treated with IL-6 (100 ng/mL). CAT activity was ascertained, as previously described.1 The sample numbers in D correspond to the sample numbers in C, with regard to the oligos used. Lane 7 indicates transfection with pCAT-Basic vector. Values are averages of 3 independent experiments, and SDs are indicated. (E) Methyl interference assay on the −65/−52 IL-6 response element to identify protein binding sites. Noncoding and coding strands were 5′ end labeled and the probe was methylated. Binding reactions were done with 50 000 cpm methylated probe and 20 μg nuclear extracts from cells treated with IL-6 for the indicated times. Resulting protein-DNA complexes were resolved on 5% nondenaturing polyacrylamide gel. After autoradiography, free and bound probes were excised and eluted from the gel. Eluted DNA was digested with piperidine for 30 minutes at 90°C and resolved on 12% DNA sequencing PAGE. F, Free Probe. Two potential binding sites were detected (indicated by open boxes). Area I covers the GGCCAATCG sequence and area II the CACTTC sequence. Schematic diagram of the JunBWT probe that was used is indicated. *Indicates G residues that are protected from cleavage based on labeling of the noncoding strand, and open stars, of the coding strand.

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