Functional Dissection of Signaling Pathways

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DESCRIPTION (provided by applicant): The cellular diversity and response to environmental changes are primarily triggered by a signaling network that is directed at the level of transcriptional regulation of most genes. A major challenge for biomedical research in the post-genomic era will be to identify and understand the mechanisms by which gene regulatory sequences coordinate gene transcription in normal and disease-affected pathways. The goal of the proposed program is to develop and make commercially available a set of validated lentiviral transcriptional reporter vectors and reporter cell lines for the most critical disease-related signal transduction pathways. These pathway-specific reporter vectors will allow researchers to study pathway mechanisms and identify changes in the mechanisms involved in the pathogenesis of human diseases. Under Phase I funding, a set of lentiviral transcriptional reporter vectors, technology for construction of transcriptional factor- specific constructs, and high complexity transcriptional reporter libraries were developed and validated for the p53 pathway in the HeLa cell model. High-throughput technology for identification of functional reporter constructs based on combination of bar-coded transcriptional reporter libraries and Affymetrix GeneChip(tm) microarrays was developed. A set of single color transcriptional reporter vectors and constructs for 15 different transcriptional factors was released as commercial products. Under Phase II funding, we propose to extend the program towards the development and commercialization of a comprehensive set of validated lentiviral reporter constructs for 25 well-characterized transcriptional factors with dual color luciferase and fluorescent reporters, covering about 30 major signal transduction pathways associated with a wide range of human diseases. The functional validation of individual constructs will be performed by screening the high complexity transcriptional reporter library against an array of different cell lines treated by different therapeutic and signal transducing agents and by knockdown of specific transcriptional factors by siRNA constructs. The established technology will be applied, in collaboration with the Cleveland Clinic Foundation, for identification of targets for drug development aimed at the restoration of suppressed p53 pathway in a set of human carcinoma cell line models. The anticipated outcomes of the proposed research and will be a commercially available set of kits comprising of lentiviral reporter constructs, a collection of stable transcriptional reporter cell lines for drug screening applications, a database of functional activity of different transcriptional response elements and transcriptional modules, and several validated anticancer drug targets. In addition, we will launch human and mouse genome-wide transcriptional reporter libraries, which will be a powerful research tool for the discovery of novel transcriptional factors associated with disease state.