Abstract 5613: A specific isoform of Gli1 binds the Gli-binding site of the c-jun and c-fos promoters

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Abstract Gli1 participates in the transcriptional regulation of c-jun, which in turn, participates in the transcriptional regulation of genes in nucleotide excision repair. The c-jun promoter and the c-fos promoter have identical Gli-binding-sites that bind Gli1. C-jun and C-fos form the transcriptionally active heterodimer Activator Protein 1, AP1. AP1, is the positive transcriptional regulator for ERCC1, and other DNA repair proteins. Gli1 is a transcription factor in the Hedgehog pathway, and there are five known isoforms of the Gli1 protein that exist in human cells. We have investigated whether there is a specific isoform of Gli1 that binds to the transcriptional regulatory sequences of c-jun and of c-fos. Detailed studies were performed in cisplatin-resistant A2780-CP70 human ovarian cancer cells. EMSA studies demonstrated the presence of a Gli protein in these cells which bind to the Gli-binding-site, as well as the consensus Gli-binding sequence. Supershift EMSA assays show that Gli1 binds the Gli binding sites of c-jun and of c-fos. Southwestern blot analyses of protein lysates from A2780-CP70 cells demonstrated that only one of the five known Gli1 protein isoforms, the 130 kDa, bind the Gli-specific binding site in the promoter of c-jun and c-fos. No Gli2 protein binds this specific binding site in the c-jun promoter, in these cells. To further confirm the 130 kDa Gli1 isoform was responsible for binding the c-jun promoter, the full-length Gli1 with a C-terminal myc tag was transfected into cells, and the protein products were assessed by immunoprecipitation and Southwestern blot analysis. The transfected full length Gli1-myc generated a 130 kDa protein that binds the Gli1-specific binding site in the promoter of c-jun. The presence of this 130 kDa Gli1 isoform was also documented in: six additional human ovarian cancer cell lines, and ten clinical ovarian tissue samples. Seven were ovarian cancer tissue and three were non-cancer ovarian tissues. The 130 kDa Gli1 isoform was present in all specimens examined, but protein levels were six-fold higher in malignant tissues. We conclude that the 130 kDa isoform of Gli1 bind the Gli-binding site in the promoters of c-jun and c-fos. Therefore, Gli1 may be the transcriptional regulator of c-jun and c-fos, and thereby regulates the transcription of ERCC1 and genes of nucleotide excision repair. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5613. doi:1538-7445.AM2012-5613