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Results
Discussion To date, only one genome-wide genetic screen has been performed in human ES alkaline phosphatase inhibitors (Chia et al., 2010). The high cost associated with an siRNA library can deter investigators from utilizing this approach to decipher human stem cell biology. In this study, we show that the piggyBac transposon can be used to conduct genetic screens in human ES cells. The ability to rapidly and cost-effectively generate a large collection of cells in which each cell has different genes mutated by simple transfection enables one to perform phenotypic-based genetic screens in human cells in a manner similar to yeast genetics. Combined with high-throughput sequencing, the genes disrupted by the piggyBac transposon can be easily identified, allowing one to study the molecular mechanisms underlying any biological process. The unique ability of the piggyBac transposon to carry large inserts offers the opportunity to conduct sophisticated insertional mutagenesis screens (Ding et al., 2005; Li et al., 2011). Here we were able to combine mutagenesis and simultaneous overexpression of a gene into a single transposon vector, performing a genetic screen in a sensitized background. Different from proteomic approaches, this sensitized screening strategy has the power to detect genes that cooperate in a biological process, e.g., blocking differentiation, regardless whether gene products physically interact with each other. With this strategy, we are able to probe the genome for genes capable of cooperating with NANOG, and by characterizing one of the clones, we were able to identify a poorly characterized gene, DENND2C, which is capable of cooperation with NANOG overexpression to block RA-induced differentiation. Although our piggyBac screen utilizes gain-of-function mutagenesis, such mutations can easily lead to the identification of loss-of-function alterations in genes in the same pathway or process, as every pathway has both positive and negative regulatory components. Indeed, we discovered that DENND2C is a negative regulator of RHOA. Concordantly, RHOA inactivation also cooperates with NANOG in blocking RA-induced differentiation. Although DENND2C was artificially overexpressed, we were able to phenocopy its effects with several different methods of RHOA inactivation, demonstrating that gain-of-function screens can be used to elucidate real biological mechanisms. Notably, both RHOA and DENND2C are expressed in human ES cells as well as differentiated cells and would have been missed as potential regulators of self-renewal and differentiation by more traditional approaches that focused on probing genes expressed uniquely in ES cells. Much of our understanding of RHOA comes from studies for its role in the cytoplasm as a key regulator of cytoskeleton dynamics (reviewed in Burridge and Doughman, 2006). Recently, RHOA has been found to be located within the nucleus, and its nuclear activity is stimulated by DNA damage (Dubash et al., 2011). We found that DENND2C negatively regulates the activity of nuclear RHOA in human ES cells. Furthermore, we discovered that nuclear RHOA is associated with DNA in both coding and non-coding regions and that this association is negatively regulated by DENND2C. In summary, forward genetic analysis by piggyBac gain of function mutagenesis has multiple advantages complimenting genomic and biochemical methods and empowers individual investigators to functionally interrogate the human ES cell genome to elucidate mechanisms underlying biology, disease, and therapeutic strategy.
Experimental Procedures
Acknowledgments
Introduction Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) offer a potentially limitless source of cells for industrial and clinical translation, and the ever-advancing field of cellular reprogramming has redefined the limits of cell plasticity (Taylor et al., 2010). Meanwhile, cell culture and production technologies are rapidly improving, and the first instances of pluripotent-cell-derived therapies have entered clinical trials (Schwartz et al., 2012).