Role of Esrp's in pluripotent stem cell biology: Cellular plasticity involving transitions between epithelial and mesenchymal states is a conserved requirement for normal embryonic development and has been implicated in the pathogenesis of human diseases such as cancer. Recently, the mesenchymal to epithelial transition (MET) was shown to be required for the reprogramming of mouse fibroblasts into induced pluripotent stem cells (iPSCs). Advancing our understanding of the mechanisms that regulate somatic cell reprogramming is essential in the development of iPSC's as safe options for regenerative medicine. The mRNA splicing factors Esrp1/2 are cell/tissue type specific master regulators of the epithelial phenotype. Consistent with this, they were shown to be among the most highly regulated genes during the MET phase of iPSC generation, the functional consequence of which was not examined. Furthermore, the Esrps are also expressed in all human and mouse embryonic stem cell lines that have been evaluated. These observations lead us to hypothesize that the Esrps expression is required in order to attain a pluripotent cell state. We also hypothesized that the Esrps may enhance cellular reprogramming and/or comprise novel pluripotency factors. Our preliminary data demonstrate a striking enhancement of iPSC generation associated with ectopic Esrp1 expression. The project described herein will further characterize this phenotype as well as test the effect of Esrp genetic deletion in stem cell biology. We will then identify the splicing program mediated by Esrps in induced and embryonic pluripotent stem cells. We will achieve these goals by carrying out the following two specific aims.
Specific Aim 1. Characterization of the role of Esrp's in somatic cell reprogramming, and maintenance of pluripotency.
Specific Aim 2. Determination of the Esrp regulated splicing program in iPS and ES cells. This project will characterize the novel role of Esrp splicing factors in iPSC's and embryonic stem cells. It will identify, in an unbiased manner and on a genome wide scale, the splicing targets of Esrps in a system with high relevance to human health. This will be one of the first efforts to identify a splicing factor specific gene expression program in stem cell biology. These results are a key requirement for future studies dedicated to mechanistically testing the functional consequences of Esrp regulated splicing events in, not only stem cell biology, but other contexts of MET such as cancer, embryonic development and organogenesis. Identifying the distinct functions of gene isoforms will be critical to advancing ou understanding of gene expression in complex organisms. Therefore projects such as this have the potential to impact biological research on an interdisciplinary scale.
Alternative splicing can produce functionally distinct products from the same gene, thereby enhancing the proteomic diversity of various cell types. We have uncovered the master regulators of an epithelial cell type- specific splicing program and determined that they also are likely to have central roles in pluripotent stem cells. Identifying a pluripotent stem cell specific splicing program will enhance our understanding of the biology that defines them and will provide new fundamental insights into using stem cells and induced pluripotent stem cells in disease modeling and regenerative medicine.