PMC:1920263 / 30434-34225 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/1920263","sourcedb":"PMC","sourceid":"1920263","source_url":"http://www.ncbi.nlm.nih.gov/pmc/1920263","text":"Sp1 and Sp3 transcription factors bind to the GC-box at position −87/−78 of the A3G promoter\nTo investigate whether the GC-box represents a binding site for the transcription factors Sp1 and Sp3, we performed EMSA analyses with nuclear extracts isolated from A3.01 T cells. As probes, we radioactively labeled the unmodified E2 sequence (nucleotides −92/−63 of the A3G promoter, probe designated APO-Sp1/3) or the same region carrying the two point mutations described above (probe designated APO-Sp1/3mut). As control, we used a commercially available Sp1 consensus oligonucleotide (Sp1cons), which is known to be recognized by Sp1 transcription factors. Four DNA–protein complexes were observed in the presence of the APO-Sp1/3 probe (Figure 6A). The upper two complexes were specific since they disappeared in the presence of a 30-fold molar excess of unlabeled APO-Sp1/3 probe (Figure 6A, lane 7). Further confirmation of the specificity of these DNA–protein complexes was demonstrated by the inability of the unlabeled APO-Sp1/3mut probe to abolish binding (Figure 6A, lanes 12 and 13). As expected, the two specific complexes were also present in the case of the Sp1cons control probe (Figure 6A, lanes 2 and 3). In contrast, none of these specific complexes was observed when the mutated probe was used (Figure 6A, lanes 10 and 11), confirming that protein binding was dependent on the identified GC-box. In order to characterize the complexes, we performed supershift experiments using Sp1- and Sp3-specific antibodies. The upper of the complexes that appeared in combination with the Sp1cons or APO-Sp1/3 probes, shifted in the presence of the Sp1 antibody (Figure 6B, lanes 3 and 7), whereas the lower complex shifted in the presence of the Sp3 antibody (Figure 6B, lanes 4 and 8). Taken together, the EMSA demonstrated that the GC-box located on the A3G core promoter serves as a binding site for Sp1 and Sp3.\nFigure 6. Sp1 and Sp3 bind to the GC-box present in the A3G promoter. (A) Nuclear extracts of A3.01 T cells were incubated with a 32P-labeled commercial Sp1 oligonucleotide probe (Sp1cons) or labeled probes homologous to the unmodified or mutated E2 region (see Figure 1) of the A3G promoter (APO-Sp1/3 and APO-Sp1/3mut). Protein–DNA complexes were separated by polyacrylamide electrophoresis and detected by autoradiography. EMSA was performed with a 1- or 30-fold molar excess of unlabeled APO-Sp1/3 probe (competitor, lanes 6 and 7) or APO-Sp1/3mut probe (competitor mut., lanes 12 and 13). (B) Sp1- and Sp3-specific antibodies were added to the EMSA reactions resulting in a supershift (ss) of the respective antibody–protein–oligo complexes (lanes 3, 4, 7, 8, 11, 12). (C) ChIP assay was performed with DNA from A3.01 T cells. Immunoprecipitation was performed with antibodies against Sp1, Sp3 or actin. PCR primer pairs specific for the A3G promoter (upper panel) or the A3G gene (lower panel) were used. As positive controls, the sheared and cross-linked DNA before the immunoprecipitation step (input) or a plasmid carrying the target sequence (plasmid) was used as template.\nTo show binding of Sp1 and Sp3 factors also in the context of the endogenous A3G promoter, a chromatin immunoprecipitation (ChIP) assay was performed. Sp1 and Sp3 antibodies, but not an actin antibody, immunoprecipitated the A3G promoter in A3.01 T cells (Figure 6C, upper panel). In contrast, no PCR signal was received with a primer pair recognizing a region in the A3G gene ∼4000 bp downstream of the Sp1/Sp3–binding site (Figure 6C, lower panel). Only the positive controls showed a DNA band, with an A3G expression plasmid or the sheared and cross-linked input DNA used as template. This demonstrates the binding of Sp1 and Sp3 transcription factors to the endogenous A3G promoter.","divisions":[{"label":"Title","span":{"begin":0,"end":92}},{"label":"Figure 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