Binding of the Sp1-p53 complex to the Sp1 site of the ERR-1 in TPA ± BI treated H9 cells.

<p>(A) Schematic illustration of the ERR-1 probe sequene: The upper probe carries the wild type sequence whereas the lower probe is mutated by 3 substituted nucleotides within the Sp1 binding site depicted in small underlined letters. (B) H9 cells were treated with TPA ± BI for the indicated times and aliquots of their nuclear extracts were tested for binding to 3′ biotin-labeled w.t. ERR-1 probe by EMSA. (C) The specificity of this protein binding to the ERR-1 probe was determined in nuclear extract of cells treated with TPA for 96 hr (i.e. at the second phase of the LTR activation). This was done by competition with 50 molar excess of unlabeled oligonucleotide carrying the w.t. or the mutated ERR-1 sequence. For additional control we employed the same excess of the oligonucleotide carrying the TRE III sequence (shown in “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#s4" target="_blank">Materials and Methods</a>” and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0029934#pone-0029934-g002" target="_blank">Figure 2A</a>) as an irrelevant competitor. (D) The proteins bound to the ERR-1 probe, were identified by supershift analysis of the nuclear extract derived from H9 cells treated with TPA for 96 hr using 1, 2 and 4 µg ofthe following antibodies: anti albumin (as an irrelevant negative control), or with anti Sp1 and the DO-1 anti p53 (which detect both the w,t. and mutant p53 proteins). A combination of anti Sp1 and anti p53 antibodies (1 and 2 µg of each of them) was employed in the last 2 lanes at the right side of the blot, whereas no antibody was employed in the control cells presented in the first lane at the left side of the blot. (E) To determine whether the Sp1-p53 complex that bound to the ERR-1 probe contained w.t. or mutant p53 protein, a similar supershift analysis was performed with pAb1620 antibody which identified only w.t. p53 and pAb240 antibody that identified only mutant p53. (F) To demonstrate the intracellular recruitment of these nuclear proteins to the ERR-1 site in LTR integrated within the cellular genome, we carried out chromatin immunoprecipitation (ChIP) analysis in the H9 cells stably transfected with LTR-Luc (H9/LTR-Luc) which were treated with TPA for 96 hr. The immunoprecipitation was performed with 2, 4 and 8 µg of each of the indicated antibodies. (G) Jurkat/LTR-Luc cells were tested for activation of their integrated LTR-Luc by 48 hr-TPA-treatment. Untreated Jurkat/LTR-Luc cells served as control. (H) Jurkat (left panel) and H9 (right panel) cells were co-transfected with the pG13-Luc reporter and BRCA1-expressing plasmid in the absence and presence of anti p53-shRNA. Cells without BRCA1 expressing plasmid served as basal control.