High resolution genomic tools for the discovery of protein function in mammalian cells

Sammanfattning: The work described in this thesis emphasizes the development of cell-based strategies with increased throughput design that enable rapid biological discovery through direct investigation of protein function. Each of the works represented in this thesis are unified by the common premise that genetic perturbations, through loss or gain-of-function, in cellbased models may serve as an entry point for assigning biological roles to uncharacterized genes and their gene products. 46 human genes were selected that encode proteins sharing a common domain with other eukaryotes and for which the molecular function is unknown. Cellular responses to overexpression were surveyed in human cell lines utilizing a panel of antibodies serving to identify distinct cellular patterns and mechanisms. The high-content capacity of reverse transfection arrays combined with the resolution of immunofluorescence microscopy allowed investigation of each protein directly within a cellular context. Three varieties of phenotypic changes associated with overexpression were pinpointed including organelle disassembly, protein translocation and post-translational modifications. As a result, new tentative biological roles were identified for two previously uncharacterized human proteins: (1) a membrane-bound O-acyltransferase protein (C3F) that, when overexpressed, disrupts Golgi and endosome integrity due likely to an ER-Golgi transport block, and (2) a tumor marker (BC2) that prompts a redistribution of a transcriptional silencing protein (BMI1) and a MAPKinase mediator (Rac1) to distinct nuclear regions that undergo chromatin compaction. In a parallel effort, loss-of-function was explored by targeted RNA interference (RNAi) for each of the 46 genes. Efficient siRNAs were selected and validated by a combinatorial approach that correctly predicts siRNAs that effectively reduce mRNA and protein levels in cultured cells. Furthermore, this method identifies proteins that have a slow turnover, a property that may weaken the value of the RNA interference method as a tool for transient studies of such genes. Using the validated set of siRNAs, the following objective aimed to identify proteins that influence cell viability and proliferation. The recently reported risk of siRNA off-targets motivated the development of a stringent validation strategy for evaluating the specificity of knockdown phenotypes for the target genes. Our strategy incorporates the use of multiple siRNAs and rescue experiments involving candidate genes delivered exogenously and containing silent mutations in the siRNA target regions. Falsely identified phenotypes were eliminated and a new role for BC2 as an anti-apoptotic gene was confirmed. BC2 has been studied in closer detail using peptide antibodies designed against the endogenous protein. Although BC-2 contains ESCRT III homology implying involvement in multivesicular body sorting and protein trafficking, insights gained from this study point to an alternate role for BC2 in the nucleus, consistent with the overexpression results reported. In addition, loss-of-function identified C3F as essential for cell proliferation. Further chemical manipulation of HeLa cells with Taxol, a stabilizer of tubulin and anti-cancer drug, revealed that C3F knockdown confers resistance to Taxol-induced apoptosis. An essential aspect of functional genomics is the comparison of gene function by homology in model organisms. The most likely worm-human orthologs for a subset of the proteins were selected by phylogenetic analysis. C.elegans genes were silenced via RNAi for lossfunctionstudies, and gene distribution profiles were analyzed using GFP strains. In addition, subcellular distributions of the human orthologs were analyzed in human cell lines. By combining a whole-organism approach with complementary studies in human cell lines, this analysis extends currently available information on the selected set of genes.