Molecular Mechanisms Defining and Driving Receptivity in Conversion of Fibroblasts to Motor Neurons

Abstract

Layers of regulation stabilize cellular identity and prevent aberrant cell-fate transitions. Cell fate conversion processes, such as the conversion of fibroblasts into motor neurons, attempt to induce a cell of one type to become a cell of another type by activating genes and gene networks associated with the desired final cell type. Cell fate conversion processes have the potential to revolutionize drug discovery and drive the development of novel cell-based therapies, but their translational potential is limited by poor conversion rates. Forced overexpression of lineage-specifying transcription factors is rarely sufficient to induce a complete change in cellular identity. We find that overexpression of known oncogenes can enhance a cell’s receptivity to conversion by increasing proliferation rates and increase conversion yields 100x, even when converting to a non-proliferative state. In this thesis, we use the model system of mouse embryonic fibroblast to motor neuron conversion to determine how oncogenic mutants of HRAS and the tumor suppressor protein p53 induce a receptive cell state and enhance conversion. We find that cells that proliferate at high rates early in conversion attain the motor neuron identity at higher rates than cells that do not proliferate as much. We isolate cells that attain high rates of proliferation and define the subcellular properties of these conversion-receptive cells. Receptive cells display more compact chromatin as proliferation destabilizes chromatin structures generally, including those that reinforce the starting cell type identity. An increase in trimethylation of H3K27, a histone mark known to induce chromatin compaction and reduce transcriptional output, supports this this compaction and is associated with a global decrease in transcription rates. Finally, we make a counter-intuitive finding that the interaction between mutant and native p53 enhances conversion beyond its role in promoting proliferation that is dependent on the presence of native p53. The p53 mutant induces accumulation of native p53 in a subpopulation of cells. We developed a tool to track p53 levels in mouse embryonic fibroblasts in order to track p53 accumulation during conversion. By developing tools to isolate cells with different conversion-receptivity during oncogene-enhanced conversion, we can characterize the subcellular features that promote conversion. We expect future cell fate conversions may be engineered to systematically guide cells through a receptive state without oncogenes.