| Summary: | It is known that the probasin (PB) promoter has the ability to dictate prostatespecific gene expression both in vitro and in vivo. Unfortunately, the regulatory factors that dictate the prostate-tissue specific gene expression are currently unknown. In this study we investigated the combination of transcription factors and proteins binding to the proximal part of the PB promoter. Using DNasel and DMS in vitro footprinting, several protected regions were identified on the proximal PB promoter (nucleotides -286 to +28 relative to the transcription start site) when nuclear extracts from LNCaP, a human prostate cancer cell line, were used. Three of the protected areas were observed only when LNCaP nuclear extracts treated with synthetic androgen (10nM R1881) were used. Two other regions, referred to as FPI and FPII, showed protection regardless of presence or absence of androgen. When DNaseI footprinting was done using other prostate and non-prostate nuclear extracts, protection of the FPII region was only seen in prostate cell lines. These androgen-independent regions were further tested for tissue and binding specificity using the electrophoresis mobility shift assay (EMSA). Eight complexes formed with the FPI probe while four complexes were observed with the FPII probe on incubation with the tested nuclear extracts. Methylation protection assays reveal that prostate cancer cell lines yield subtly different protection patterns for some of the protein complexes formed with non-prostate derived cell lines, suggesting the presence of prostate-enriched or -exclusive proteins. In an attempt to identify the protein(s) binding to the FPII region, transcription factor database searches were used. Two potential candidates, c-Myb and NF-I, were identified. Binding assays, including supershift experiments revealed the participation of NF-I or a closely related protein, although other unknown proteins are also involved. In transient transfection studies, site directed mutagenesis of the putative NF-I site within FPII resulted in a significant reduction in PB promoter activity compared to the wild type promoter. Defining the DNA and protein components that dictate prostate-specific expression of the probasin promoter in an androgen-independent manner would provide a strong basis for the design and development of a gene therapy for systemic treatment of androgen independent prostate cancer. === Medicine, Faculty of === Pathology and Laboratory Medicine, Department of === Graduate
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