Multicellular organisms begin as a single cell, executing an intricate choreography of genetic and epigenetic regulation that results in the formation of a complete organism. As pluripotent cells divide and specialize, their potential to be converted to other cell types becomes increasingly restricted. The manipulation of this process to convert easily obtained, easily grown and self-renewing cell types, such as fibroblasts, is a much-studied and sought-after goal for the potential therapies it envisions, including the creation of patient-specific neurons to use as a replacement for cells damaged in stroke, spinal injury, or neurodegenerative disease. The conversion of one cell fate to another, or cellular reprogramming, is dependent on cellular context and involves both genetic and epigenetic changes. While some efforts have been fruitful in directly converting one cell type into another by overexpressing key cell-fate specifying transcription factors, the mechanism underlying the context dependency and the characteristics of the reprogrammed cells remain largely unknown and hamper further progress in the field.
I aim i n this proposal to combine the power of genetics in Caenorhabditis elegans with a reprogramming assay with proven effectiveness, which was developed in our lab, to elucidate the mechanism and characteristics of reprogramming. Using this assay, our lab has shown recently that the removal of a single chromatin-related gene can broaden the ability of transcription factor overexpression to reprogram cells. This evidence led me to perform a genetic screen for mutants that also allow broadened reprogramming. I have thus far isolated for 40 mutants with these characteristics, to which I will in aim 1 use a whole-genome sequencing strategy pioneered in our lab to efficiently identify the phenotype-causing mutations. I will then in aim 2 allow the gene identities to guide phenotypic characterization, including expression pattern, extent and specificity of reprogramming, biochemical and genetic interaction partners, and the epigenetic characteristics of reprogrammed cells. These data will allow me to form a coherent model of cell fate restriction and reprogramming, which will further both basic and applied science fields.
This proposal aims to understand why cells lose the potential to adopt multiple different cell fates as they differentiate and specialize during development. The elucidation of mechanisms underlying this fascinating yet poorly understood process will help to understand not only fundamental aspects of metazoan development but will also offer insight into how this process may be manipulated for therapeutic reprogramming, which envisions the creation of patient-specific tissues by reprogramming easily obtained, easily grown and self-renewing cell types, such as fibroblasts, into the desired tissue type as a means to replace non-regenerative tissue damaged by injury or disease. Without a fundamental understanding of the mechanisms and characteristics of reprogramming, it will be difficult to attain such a highly vaunted goal;this proposal aims to help understand this phenomenon on a fundamental level.
